Methods and compounds for prevention and treatment of elevated intraocular pressure and related conditions

ABSTRACT

A GPCR-like protein is described, as well as inhibitory/antagonistic compounds and compositions comprising such inhibitors/antagonists of the protein. Such compounds may be used for treating elevated intraocular pressure and conditions associated with elevated intraocular pressure, such as glaucoma.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisionalapplication No. 60/367,513 filed Mar. 27, 2002.

FIELD OF THE INVENTION

[0002] The invention relates to methods and compounds for the preventionand/or treatment of ocular hypertension or elevated intraocularpressure, and related conditions such as glaucoma.

BACKGROUND OF THE INVENTION

[0003] Glaucoma and Intraocular Pressure

[0004] Glaucoma is characterized by optic nerve head excavation whichcan lead to loss of peripheral vision and sometimes loss of centralvision. Glaucoma is the second leading cause of vision loss worldwide;an estimated 66.8 million people will have primary glaucoma and 6.7million will be bilaterally blind due to this disorder. In the UnitedStates, glaucoma is the second leading cause of permanent blindness andthe leading cause among African Americans. While glaucoma is treatableand vision loss can be prevented, once vision loss occurs it isirreversible. Elevated intraocular pressure (IOP) is a major risk factorfor the development of glaucoma, but it is not found in all patientswith the disease (Sommer, A. et al., Arch. Ophthalmol. 109: 1090-1095(1991)). Glaucoma associated with elevated IOP is divided into threemajor categories: open angle, closed angle and developmental. Each ofthese categories is further divided into primary and secondary forms,and by the age of onset. Drugs that lower IOP, such as latanoprost,unoprost, timolol which target prostaglandin F_(2α) and β₂ adrenergicreceptors are being currently used to prevent and delay oculardegeneration in glaucomatous patients.

[0005] There is therefore a continued need to identify and characterizetargets and in turn products for the prevention and treatment of oculardisorders, such as glaucoma.

SUMMARY OF THE INVENTION

[0006] The invention relates to R-14 nucleic acids and polypeptides andcompounds capable of lowering intraocular pressure and uses thereof.

[0007] Accordingly, in an aspect, the invention provides a substantiallypure peptide compound of Formula I:

Z₁-X₁—X₂—X₃—X₄—-X₅—X₆—X₇—X₈-Z₂  I

[0008] wherein:

[0009] X₁ is selected from the group consisting of Phe, His, Ile andrelated amino acids;

[0010] X₂ is selected from the group consisting of Ser, Ile, Phe andrelated amino acids

[0011] X₃ is selected from the group consisting of Leu, Ile, Asp andrelated amino acids

[0012] X₄ is selected from the group consisting of Thr, Cys, Ser andrelated amino acids possessing side chains containing sulfhydryl,hydroxyl or H-bond forming groups;

[0013] X₅ is selected from the group consisting of Gln, Ser, Thr andrelated amino acids;

[0014] X₆ is selected from the group consisting of Lys, Pro, Glu andrelated amino acids;

[0015] X₇ is selected from the group consisting of Tyr, Leu, Cys andrelated amino acids;

[0016] X₈ is selected from the group consisting of Cys, Arg, Trp andrelated amino acids;

[0017] Z₁ is an N-terminal group of the formula H₂N—, RHN— or, RRN—;

[0018] Z₂ is a C-terminal group of the formula —C(O)OH, —C(O)R, —C(O)OR,—C(O)NHR, —C(O)NRR;

[0019] R at each occurrence is independently selected from (C₁-C₆)alkyl, (C₁-C₆) alkenyl, (C₁-C₆) alkynyl, substituted (C₁-C₆) alkyl,substituted (C₁-C₆) alkenyl, or substituted (C₁-C₆) alkynyl; and

[0020] “-” is a covalent linkage.

[0021] The invention further provides a substantially pure syntheticpeptide compound or recombinant peptide compound, said compound having adomain of Formula II:

—X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈—  II

[0022] wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈ and “-” are as definedabove.

[0023] In embodiments, the compound is selected from the groupconsisting of: FSLTQKYC; HIICSPLR; and IFDSTECW. In embodiments, thedomain is selected from the group consisting of: FSLTQKYC; HIICSPLR; andIFDSTECW.

[0024] The invention further provides a method of lowering intraocularpressure in a subject, said method comprising administering to saidsubject an effective amount of the above-mentioned compound.

[0025] The invention further provides a method of treating in a subjecta condition associated with elevated intraocular pressure, said methodcomprising administering to said subject an effective amount of theabove-mentioned compound.

[0026] In embodiments, the methods comprise administering said compoundto an eye of said subject.

[0027] The invention further provides a pharmaceutical compositioncomprising the above-mentioned compound in admixture with apharmaceutically acceptable carrier. In an embodiment, the compositionis formulated for administration to an eye of a subject.

[0028] The invention further provides a commercial package comprisingthe above-mentioned compound together with instructions for its use. Inembodiments, the use is selected from the group consisting of: (a)lowering intraocular pressure in a subject; and (b) treating a conditionassociated with elevated intraocular pressure.

[0029] The invention further provides an isolated nucleic acidcomprising a sequence that encodes the above-mentioned domain. Theinvention further provides a vector comprising the nucleic acidoperably-linked to a transcriptional regulatory element. The inventionfurther provides a host cell comprising the vector.

[0030] The invention further provides a method of producing theabove-mentioned peptide compound, comprising culturing theabove-mentioned host cell under conditions permitting expression of thepeptide compound.

[0031] The invention further provides an isolated nucleic acidcomprising a sequence that encodes a polypeptide comprising at least 278amino acids of SEQ ID NO:2. In an embodiment, the polypeptide comprisesSEQ ID NO:2. In an embodiment, the nucleic acid comprises SEQ ID NO:1 ora sequence substantially identical thereto.

[0032] The invention further provides a substantially pure polypeptidecomprising at least 278, consecutive amino acids of SEQ ID NO:2. In afurther embodiment, the polypeptide comprises SEQ ID NO:2.

[0033] The invention further provides a vector comprising theabove-mentioned nucleic acid operably linked to a transcriptionalregulatory element. The invention further provides a host cellcomprising the above-mentioned vector.

[0034] The invention further provides a method of producing theabove-mentioned polypeptide, comprising culturing the above-mentionedhost cell under conditions permitting the expression of the polypeptide.

[0035] The invention further provides a method for identifying acompound for: (i) lowering intraocular pressure in a subject; (ii)treating a condition associated with elevated intraocular pressure; or(iii) both (i) and (ii); said method comprising: (a) providing a cellexpressing an R-14; (b) contacting the cell with a test compound; anddetermining whether R-14 activity is decreased in the presence of saidtest compound, said decrease in activity being an indication that saidcompound may be useful for: (i) lowering intraocular pressure in asubject; (ii) treating a condition associated with elevated intraocularpressure; or (iii) both (i) and (ii). In an embodiment, said decrease inactivity is an indication that said compound is an R-14 antagonist. Inan embodiment, R-14 comprises a polypeptide comprising SEQ ID NO:2, anactive fragment thereof, or a sequence substantially identical thereto.

[0036] The invention further provides a use of the above-mentionedcompound for: (a) lowering intraocular pressure in a subject; (b)treating a condition associated with elevated intraocular pressure; or(c) both (a) and (b).

[0037] The invention further provides a use of the above-mentionedcompound or the above-mentioned composition for the preparation of amedicament. In an embodiment, the medicament is for: (a) loweringintraocular pressure in a subject; (b) treating a condition associatedwith elevated intraocular pressure; or (c) both (a) and (b).

[0038] In an embodiment, the above-mentioned condition is glaucoma.

[0039] In an embodiment, the above-mentioned subject is a mammal, in afurther embodiment, a human.

[0040] In further embodiments, Z₁ is selected from the group consistingof a proton, a sequence of 1-3 amino acids, or a blocking group such asa carbamate group, an acyl group composed of a hydrophobic moiety suchas cyclohexyl, phenyl, benzyl, short chain linear and branched alkylgroups of 1-8 carbons. In further embodiments Z₂ is selected from thegroup consisting of proton, NH₂, 1-3 amino acids as well as arylalkylamines such as benzylamine, phenylethylamine, phenylpropylamine, andaliphatic amines possessing short chain linear and branched alkyl groupsof 1 to 8 carbons.

[0041] In another aspect, the invention provides a method forformulating a medicament, the method comprising admixing a compound ofthe invention with a pharmaceutically acceptable carrier.

[0042] In another aspect, the invention provides a method of loweringintraocular pressure in a subject, the method comprising inhibitingexpression and/or activity of an R-14 protein in the subject. In anembodiment, the R-14 protein comprises a polypeptide selected from thegroup consisting of:

[0043] (a) the polypeptide of SEQ ID. NO. 2; and

[0044] (b) a polypeptide encoded by a first nucleic acid that issubstantially identical to a second nucleic acid capable of encoding thepolypeptide of SEQ ID NO. 2. In an embodiment, the second nucleic acidis as set forth in SEQ ID NO. 1.

[0045] In an embodiment, the above-noted method comprises administeringto the subject an effective amount of an agent capable of modulatingR-14 activity. In an embodiment, the agent is a compound of theinvention. In a further embodiment, the method comprises administeringto the subject a therapeutically-effective dose of the above-mentionedcomposition. In an embodiment, the dose is of about 0.001 mg to of about100 mg.

[0046] The invention further provides a method of lowering intraocularpressure in a subject, the method comprising administering to thesubject an effective amount of a compound of the invention. In anembodiment, the method comprises administering to the subject atherapeutically-effective dose of the above-mentioned composition. In anembodiment, the dose is of about 0.001 mg to of about 100 mg.

[0047] The invention further provides a method of preventing or treatingin a subject a condition associated with elevated intraocular pressure,the method comprising inhibiting expression and/or activity of an R-14protein in the subject. In an embodiment, the R-14 protein comprises apolypeptide selected from the group consisting of:

[0048] (a) the polypeptide of SEQ ID NO. 2; and

[0049] (b) a polypeptide encoded by a first nucleic acid that issubstantially identical to a second nucleic acid capable of encoding thepolypeptide of SEQ ID NO. 2. In an embodiment, the second nucleic acidis as set forth in SEQ ID NO. 1.

[0050] In an embodiment, the method comprises administering to thesubject an effective amount of an agent capable of modulating R-14activity. In an embodiment, the agent is a compound of the invention. Ina further embodiment, the method comprises administering to the subjecta therapeutically-effective dose of the above-mentioned composition. Inan embodiment, the dose is of about 0.001 mg to of about 100 mg.

[0051] The invention further provides a method of preventing or treatingin a subject a condition associated with elevated intraocular pressure,the method comprising administering to the subject an effective amountof a compound of the invention. In a further embodiment, the methodcomprises administering to the subject a therapeutically-effective doseof the above-mentioned composition. In an embodiment, the dose is ofabout 0.001 mg to of about 100 mg.

[0052] The invention further provides a commercial package comprising anagent capable of modulating R-14 activity together with instructionsfor:

[0053] (a) lowering intraocular pressure in a subject;

[0054] (b) preventing or treating a condition associated with elevatedintraocular pressure; or

[0055] (c) both (a) and (b).

[0056] In an embodiment, the R-14 protein comprises a polypeptideselected from the group consisting of:

[0057] (i) the polypeptide of SEQ ID NO. 2; and

[0058] (ii) a polypeptide encoded by a first nucleic acid that issubstantially identical to a second nucleic acid capable of encoding thepolypeptide of SEQ ID NO. 2.

[0059] The invention further provides a method for identifying and/orcharacterizing a compound for lowering intraocular pressure, the methodcomprising assaying the activity of an R-14 in the presence of a testcompound, to identify a compound that acts as an R-14 antagonist,wherein antagonist activity is indicative that the test compound may beuseful for lowering intraocular pressure.

[0060] The invention further provides a method for identifying and/orcharacterizing a compound for lowering intraocular pressure, the methodcomprising:

[0061] (a) contacting a test compound with a host cell expressing apolypeptide selected from the group consisting of:

[0062] (i) the polypeptide of SEQ ID NO. 2; and

[0063] (ii) a polypeptide encoded by a first nucleic acid that issubstantially identical to a second nucleic acid capable of encoding thepolypeptide of SEQ ID NO. 2; and

[0064] (b) assaying activity of an R-14 in the presence of the testcompound, to identify a compound that acts as an R-14 antagonist,wherein antagonist activity is indicative that the test compound may beuseful for lowering intraocular pressure. In an embodiment, the compoundmay be used for the prevention and/or treatment of a conditionassociated with intraocular pressure.

[0065] In an embodiment, the above-mentioned subject is a mammal, in afurther embodiment, a human.

[0066] In an embodiment, the above-mentioned condition is glaucoma.

BRIEF DESCRIPTION OF THE DRAWINGS

[0067]FIG. 1: (A) Agarose gel electrophoretic resolution of ˜960 bpproduct (arrow) obtained by PCR amplification as described in Example 1.−Bac: No plasmid DNA; +Bac: contains plasmid DNA (0.1 μg); MW std: λHind III digest. (B). The nucleotide sequence of the R-14 reading frame(SEQ ID NO:1). (C) R-14 polypeptide sequence (SEQ ID NO:2).

[0068]FIG. 2: Agarose gel electrophoretic analysis of RT-PCR productsfor analysis of R-14 expression as described in Example 2. Iris: samplederived from Iris tissue; HTM: sample derived from human trabecularmeshwork tissue.

[0069]FIG. 3: Immunoblot analysis of expression of cloned human R-14receptor in human cells as described in Example 3. Arrow shows R-14immunoreactive band.

[0070]FIG. 4: Graphical results of identification and characterizationof inhibitors of R-14 protein as described in Example 4.

[0071]FIG. 5: Graphical results of comparison of the efficacy of peptide1405 with Latanoprost and Timolol as described in Example 5.

[0072]FIG. 6: Graphical results of analysis of selectivity of peptides1402 and 1405 as described in Example 6.

[0073]FIG. 7: Graphical results of effect of peptides 1402 (A) and 1405(B) on intraocular pressure in rabbits, as described in Example 7.

[0074] Other objects, advantages and features of the present inventionwill become more apparent upon reading of the following non-restrictivedescription of preferred embodiments with reference to the accompanyingdrawings, which is exemplary and should not be interpreted as limitingthe scope of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0075] Identification of Novel GPCRs

[0076] Use of low stringency hybridization, differential display,microarrays, subtractive hybridization and other techniques followed bycloning of differentially expressed genes in tissues have disclosedseveral new cDNAs which could potentially code for new G protein-coupledreceptors. Following the elucidation of the draft sequence of the humangenome (Venter, J C. et al., Science. 291: 1304-1351 (2001);International Human Genome Sequencing Consortium. Nature. 409: 860-921(2001)), many GPCR-like sequences have been identified based on theircanonical seven transmembrane topology as well as conserved proteinmotifs (Howard, D A et al., 2001. Trends Pharmacol. Sci. 22(3):132-140). These unique GPCR-like sequences number ˜1000 of whichnonolfactory GPCR sequences are estimated to be ˜616.

[0077] Gonzalez et al. (Gonzalez, P. et al., 2000. Invest. Ophthalmol.Vis. Sci. 41: 3678-3673), carried out single pass sequencing of 1060cDNA clones isolated from human trabecular meshwork, including asequence having homology to MAS-related G-protein coupled receptor(denoted as HTMI-0025, and corresponding to sequences Hs11_(—)9464 andHs11_(—)24438 found in the human chromosome 11 working draft sequence).

[0078] Dong et al. (Dong, X. et al. 2001. Cell 106:619-632) clonedseveral mas-related cDNAs from mouse embryos lacking neurogenin gene,with subsequent searching among human genome sequences revealing fourrelated genes (called Hs_mrgX1-4).

[0079] When the physiological ligand(s) of a GPCR is not known, then itis termed an orphan GPCR. In order to find ligands of these orphanGCPRs, random libraries containing natural peptides isolated from thetissues or those containing small molecules produced by combinatorialchemistry or from natural sources are screened in cell-based and invitro assays. Selective modulators of the orphan GPCR can be used toassess its function in physiology and pathophysiology using appropriateanimal models. Finding a function of the orphan GPCR is a first steptowards analyzing its potential as a new drug target.

[0080] The studies described herein relate to a novel GPCR proteinexpressed in trabecular meshwork, nucleic acids capable of encoding it,and peptide compounds capable of modulating phenomena in ocular tissue,notably intraocular pressure.

[0081] In one aspect, the invention provides an isolated a GPCR-likereading frame, which is capable of expressing mRNA and protein, and isassociated with a role in ocular outflow in animals. The inventionfurther provides three antagonists of the GPCR which could be used forlowering intraocular pressure, and thus for prevention/treatment ofdisorders associated with elevated intraocular pressure, such asglaucoma and related conditions.

[0082] As described herein, applicants have cloned a genomic DNAfragment of 0.97 Kb in length from a BAC clone and have shown that itcontained a single exon encoding a 322 amino acid long protein. Thisprotein is termed as R-14. From in silico analysis, R-14 appears to bean integral membrane protein containing seven prominent transmembranedomains and many protein motifs of G protein-coupled receptors. Byemploying RT-PCR, R-14 mRNA is shown to be expressed in human trabecularmeshwork, but not in another ocular tissue, iris. R-14 reading frame wascloned into mammalian expression vectors and transiently as well asstably expressed in HEK293 cells. Using these cells, a protein of 40 kDawas identified to be encoded by the recombinant R-14 gene. Furthermore,using selective peptidic ligands, applicants have shown that inhibitionof R-14 receptor resulted in reduction of basal cyclic AMP synthesis,but not basal phosphoinositide levels, in porcine trabecular meshworkand most importantly, reduction of basal intraocular pressure inrabbits, adult pig eyes and anesthetized piglets. Based on thisinformation, R-14 receptor is identified as a useful target forscreening for ocular hypotensive drugs, which may be useful fortreatment of conditions associated with elevated intraocular pressuresuch as glaucoma and related conditions.

[0083] Furthermore, compositions containing selective inhibitors of R-14receptor are described which could be potentially used as ocularhypotensive drugs and glaucoma therapeutics or as lead compounds towardsdevelopment of such drugs. Bioassays in which host cells (e.g. mammaliancells) containing recombinantly expressed R-14 are described which canbe used to screen chemical compound libraries to identify lead compoundsfor providing R-14 ligands which in turn can be optimized into ocularhypotensive drugs and glaucoma therapeutics.

[0084] Definitions

[0085] For convenience, the meaning of certain terms and phrasesemployed in the specification, examples, and appended claims areprovided below.

[0086] The term “agonist”, as used herein, is meant to refer to an agentthat potentiates at least one aspect of R-14 bioactivity. R-14bioactivity can be increased for example, by stimulating the wild-typeactivity and signal transduction, or enable the wild type R-14 proteinto interact more efficiently with other proteins which are involved insignal transduction cascade.

[0087] “Antagonist” as used herein is meant to refer to an agent thatinhibits at least one R-14 bioactivity. An R-14 antagonist can be acompound which inhibits or decreases the interaction between a R-14protein and another molecule, or decreases the synthesis and expressionof R-14 polypeptide or inhibits the bioactivity of R-14 molecule. Theantagonist can be a nucleic acid molecule, such as a dominant negativeform of R-14, an R-14 antisense molecule, a ribozyme capable ofspecifically interacting with R-14 mRNA, or molecules which bind to aR-14 polypeptide e.g. peptides, antibodies, small molecules.

[0088] An “agent capable of modulating R-14 activity” refers to anycompound which when introduced into a system comprising an R-14 protein,is capable of altering at least one aspect of R-14 activity or function.Such an agent may be a ligand of an R-14 protein, in further embodimentsan agonist or antagonist as defined above. Such an agent may actdirectly on an R-14 protein or indirectly by modulating a process oractivity which subsequently results in the modulation of R-14 activity.In certain systems (e.g. in vivo), such an agent may be a prodrug whichis metabolised to an active form at or prior to its arrival at the siteof action. Examples of R-14 activity are noted below.

[0089] The term “amino acid” as used herein includes both L and Disomers of the naturally occurring amino acids (Table 1) as well asother nonproteinaceous amino acids used in peptide chemistry to preparesynthetic analogs of peptides. Examples of naturally-occurring aminoacids are glycine, alanine, valine, leucine, isoleucine, serine,threonine, etc. whereas nonproteinaceous amino acids are norleucine,norvaline, cyclohexyl alanine, biphenyl alanine, homophenyl alanine,naphthyl alanine, pyridyl alanine, phenyl alanines substituted at theortho, para and meta positions with alkoxy, halogen or nitro groups etc.These compounds are known to persons versed in the art of peptidechemistry. TABLE 1 Common notations of amino acids (L-amino acids bycapital letters and D-amino acids by small letters are represented byconvention; glycine does not have L/D forms) 3-Letter 1-Letter Full NameCode Code Aspartic Asp D/d Acid Glutamic Glu E/e Acid Lysine Lys K/kArginine Arg R/r Histidine His H/h Tyrosine Tyr Y/y Cysteine Cys C/cAsparagine Asn N/n Glutamine Gln Q/q Serine Ser S/s Threonine Thr T/tGlycine Gly G Alanine Ala A/a Valine Val V/v Leucine Leu L/l IsoleucineIle I/I Methionine Met M/m Proline Pro P/p Phenyl- Phe F/f alanineTryptophan Trp W/w

[0090] “Biological activity” or “bioactivity” or “activity” or“biological function”, which are used interchangeably, for the purposesherein means a function that is directly or indirectly performed by anR-14 polypeptide, or by any fragment thereof. In this instance,biological activities of R-14 include binding to another molecule,interaction with other proteins, alterations in signal transduction suchas guanine nucleotide binding by G_(α) proteins, calcium fluxes, cAMPsynthesis, inositol phosphate synthesis, internalization of R-14polypeptide, association with other intracellular proteins or coatedpits in cell membrane, alterations in intraocular pressure, elevation ofaqueous humor transit through the Schlemm's canal etc.

[0091] “Cells”, “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular cell but to all its progeny. Also within the scope ofthe term are cells of mammalian (e.g. human), amphibian, fungal (e.g.yeast), and bacterial (e.g. E. coli) origin.

[0092] A “fusion polypeptide” can be represented by the general formulaX-(R-14)-Y, wherein R-14 represents a portion of the polypeptide whichis derived from a R-14 polypeptide, and X and Y are amino acid sequencesand are independently present or absent in the fusion peptide.

[0093] As used herein, the term “nucleic acid” refers to polynucleotidesor oligonucleotides such as deoxyribonucleic acid (DNA), and, whereappropriate, ribonucleic acid (RNA).

[0094] The terms “protein” and “polypeptide” are used interchangeablyherein when referring to a gene product.

[0095] The term “R-14 nucleic acid” refers to a nucleic acid capable ofencoding an R-14 protein, such as nucleic acids having SEQ ID NO. 1, aswell as fragments thereof, and sequences substantially identicalthereto.

[0096] The terms “R-14 polypeptide” and “R-14 protein” are intended toencompass polypeptides comprising the amino acid sequence shown as SEQID NO:2 or fragments, variants and homologs thereof.

[0097] “Homology” and “homologous” refers to sequence similarity betweentwo peptides or two nucleic acid molecules. Homology can be determinedby comparing each position in the aligned sequences. A degree ofhomology between nucleic acid or between amino acid sequences is afunction of the number of identical or matching nucleotides or aminoacids at positions shared by the sequences. An “unrelated” or“non-homologous” sequence shares less than 40% identity, thoughpreferably less than about 25% identity, with SEQ ID Nos:1 or 2 of thepresent invention.

[0098] Substantially complementary nucleic acids are nucleic acids inwhich the “complement” of one molecule is substantially identical to theother molecule. Two nucleic acid or protein sequences are considered“substantially identical” if, when optimally aligned, they share atleast about 70% sequence identity. In alternative embodiments, sequenceidentity may for example be at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95%. Optimal alignment of sequences forcomparisons of identity may be conducted using a variety of algorithms,such as the local homology algorithm of Smith and Waterman,1981, Adv.Appl. Math 2: 482, the homology alignment algorithm of Needleman andWunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and thecomputerised implementations of these algorithms (such as GAP, BESTFIT,FASTA and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, Madison, Wis., U.S.A.). Sequence identity may also bedetermined using the BLAST algorithm, described in Altschul et al.,1990, J. Mol. Biol. 215:403-10 (using the published default settings).Software for performing BLAST analysis may be available through theNational Center for Biotechnology Information (through the internet athttp://www.ncbi.nlm.nih.gov/). The BLAST algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence that either match or satisfysome positive-valued threshold score T when aligned with, a word of thesame length in a database sequence. T is referred to as theneighbourhood word score threshold. Initial neighbourhood word hits actas seeds for initiating searches to find longer HSPs. The word hits areextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extension of the word hitsin each direction is halted when the following parameters are met: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTprogram may use as defaults a word length (W) of 11, the BLOSUM62scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of bothstrands. One measure of the statistical similarity between two sequencesusing the BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. In alternativeembodiments of the invention, nucleotide or amino acid sequences areconsidered substantially identical if the smallest sum probability in acomparison of the test sequences is less than about 1, preferably lessthan about 0.1, more preferably less than about 0.01, and mostpreferably less than about 0.001.

[0099] An alternative indication that two nucleic acid sequences aresubstantially complementary is that the two sequences hybridize to eachother under moderately stringent, or preferably stringent, conditions.Hybridization to filter-bound sequences under moderately stringentconditions may, for example, be performed in 0.5 M NaHPO₄, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1%SDS at 42° C. (see Ausubel, et al. (eds), 1989, Current Protocols inMolecular Biology, Vol. 1, Green Publishing Associates, Inc., and JohnWiley & Sons, Inc., New York, at p. 2.10.3). Alternatively,hybridization to filter-bound sequences under stringent conditions may,for example, be performed in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65° C.,and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel, et al. (eds),1989, supra). Hybridization conditions may be modified in accordancewith known methods depending on the sequence of interest (see Tijssen,1993, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York). Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point forthe specific sequence at a defined ionic strength and pH.

[0100] The term “interact” as used herein is meant to include detectablerelationships or associations (e.g. biochemical interactions) betweenmolecules, such as interaction between protein-protein, protein-nucleicacid, nucleic acid-nucleic acid, and protein-small molecule or nucleicacid-small molecule in nature.

[0101] The term “modulation” as used herein refers to both upregulation(i.e., activation or stimulation (e.g., by agonizing or potentiating))and downregulation (i.e. inhibition or suppression (e.g., byantagonizing, decreasing or inhibiting)).

[0102] The term “peptide” is intended to mean a linear polymercontaining at least 2 amino acids to a maximum of 50 amino acids. Infurther embodiments the peptide contains 2 to 25 amino acids, 2 to 20amino acids, 2 to 15 amino acids, 2 to 10 amino acids or 8 amino acids.The amino acids can be naturally-occuring or synthetically-derivedmolecules. Examples of such molecules are L-amino acids, D-amino acids,and synthetic analogues of natural amino acids including but not limitedto nonproteinaceous amino acids.

[0103] The term “peptidomimetic” refers to a molecule that mimics thestructural and/or functional features of a peptide. Persons skilled inthe art use variety of methods to derive peptidomimetics of a peptide:substitutions of individual amino acids with synthetic chemicalentities, nonproteinaceous amino acid analogues, deletions, additions ofamino acids, replacing one or more of amino acids in the peptide withscaffolds such as beta turn mimetics, or with known pharmacophores. Adescription of the general methods are given in Peptidomimetic protocols(Methods in molecular medicine Vol. 23) W. M. Kazmierski (ed.), HumanaPress and Advances in Amino Acid Mimetics and Peptidomimetics, Vols. 1 &2 A. Abell (Ed).

[0104] The term “recombinant protein” refers to a polypeptide of thepresent invention which is produced by recombinant DNA techniques,wherein generally, DNA encoding a R-14 polypeptide is inserted into asuitable expression vector which is in turn used to transform a hostcell to produce the heterologous protein. “Recombinant” means thatsomething has been recombined, so that when made in reference to anucleic acid construct the term refers to a molecule that is comprisedof nucleic acid sequences that are joined together or produced by meansof molecular biological techniques. The term “recombinant” when made inreference to a protein or a polypeptide refers to a protein orpolypeptide molecule which is expressed using a recombinant nucleic acidconstruct created by means of molecular biological techniques. The term“recombinant” when made in reference to genetic composition refers to agamete or progeny or cell or genome with new combinations of allelesthat did not occur in the parental genomes. Recombinant nucleic acidconstructs may include a nucleotide sequence which is ligated to, or ismanipulated to become ligated to, a nucleic acid sequence to which it isnot ligated in nature, or to which it is ligated at a different locationin nature. Referring to a nucleic acid construct as ‘recombinant’therefore indicates that the nucleic acid molecule has been manipulatedusing genetic engineering, i.e. by human intervention. Recombinantnucleic acid constructs may for example be introduced into a host cellby transformation. Such recombinant nucleic acid constructs may includesequences derived from the same host cell species or from different hostcell species, which have been isolated and reintroduced into cells ofthe host species. Recombinant nucleic acid construct sequences maybecome integrated into a host cell genome, either as a result of theoriginal transformation of the host cells, or as the result ofsubsequent recombination and/or repair events.

[0105] The term “vector” refers to a nucleic acid molecule, which iscapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Preferred vectors are thosecapable of autonomous replication and/or expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”.

[0106] The recombinant expression vector of the present invention can beconstructed by standard techniques known to one of ordinary skill in theart and found, for example, in Sambrook et al. (1989) in MolecularCloning: A Laboratory Manual. A variety of strategies are available forligating fragments of DNA, the choice of which depends on the nature ofthe termini of the DNA fragments and can be readily determined bypersons skilled in the art. The vectors of the present invention mayalso contain other sequence elements to facilitate vector propagationand selection in bacteria and host cells. In addition, the vectors ofthe present invention may comprise a sequence of nucleotides for one ormore restriction endonuclease sites. Coding sequences such as forselectable markers and reporter genes are well known to persons skilledin the art.

[0107] A recombinant expression vector comprising a nucleic acidsequence of the present invention may be introduced into a host cell,which may include a living cell capable of expressing the protein codingregion from the defined recombinant expression vector. The living cellmay include both a cultured cell and a cell within a living organism.Accordingly, the invention also provides host cells containing therecombinant expression vectors of the invention. The terms “host cell”and “recombinant host cell” are used interchangeably herein. Such termsrefer not only to the particular subject cell but to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

[0108] Vector DNA can be introduced into cells via conventionaltransformation or transfection techniques. The terms “transformation”and “transfection” refer to techniques for introducing foreign nucleicacid into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection,electroporation, microinjection and viral-mediated transfection.Suitable methods for transforming or transfecting host cells can forexample be found in Sambrook et al. (Molecular Cloning: A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), andother laboratory manuals. Methods for introducing DNA into mammaliancells in vivo are also known, and may be used to deliver the vector DNAof the invention to a subject for gene therapy for lowering intraocularpressure and/or for treating associated conditions, such as glaucoma.

[0109] “Transcriptional regulatory sequence/element” is a generic termthat refers to DNA sequences, such as initiation and terminationsignals, enhancers, and promoters, splicing signals, polyadenylationsignals which induce or control transcription of protein codingsequences with which they are operably linked. In the present invention,transcription of one of the R-14 genes is under the control of apromoter sequence which controls the expression of the recombinant genein a cell-type. A first nucleic acid sequence is “operably-linked” witha second nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably-linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequences. Generally, operably-linked DNA sequences arecontiguous and, where necessary to join two protein coding regions, inreading frame. However, since for example enhancers generally functionwhen separated from the promoters by several kilobases and intronicsequences may be of variable lengths, some polynucleotide elements maybe operably-linked but not contiguous.

[0110] As used herein, the term “transfection” means the introduction ofa nucleic acid, e.g., via an expression vector, into a recipient cell bynucleic acid-mediated gene transfer.

[0111] A cell (e.g. a host cell or indicator cell), tissue, organ, ororganism into which has been introduced a foreign nucleic acid (e.g.exogenous or heterologous DNA [e.g. a DNA construct]), is considered“transformed”, “transfected”, or “transgenic”. A transgenic ortransformed cell or organism also includes progeny of the cell ororganism and progeny produced from a breeding program employing atransgenic organism as a parent and exhibiting an altered phenotyperesulting from the presence of a recombinant nucleic acid construct. Atransgenic organism is therefore an organism that has been transformedwith a heterologous nucleic acid, or the progeny of such an organismthat includes the transgene. The introduced DNA may be integrated intochromosomal DNA of the cell's genome, or alternatively may be maintainedepisomally (e.g. on a plasmid). Methods of transfection are well knownin the art (Sambrook et al., 1989, supra; Ausubel et al., 1994 supra).

[0112] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (such as resistance to antibiotics) may beintroduced into the host cells along with the gene of interest. As usedherein, the term “selectable marker” is used broadly to refer to markerswhich confer an identifiable trait to the indicator cell. Non-limitingexample of selectable markers include markers affecting viability,metabolism, proliferation, morphology and the like. Preferred selectablemarkers include those that confer resistance to drugs, such as G418,hygromycin and methotrexate. Nucleic acids encoding a selectable markermay be introduced into a host cell on the same vector as that encodingthe peptide compound or may be introduced on a separate vector. Cellsstably transfected with the introduced nucleic acid may be identified bydrug selection (cells that have incorporated the selectable marker genewill survive, while the other cells die).

[0113] A compound is “substantially pure” when it is separated from thecomponents that naturally accompany it. Typically, a compound issubstantially pure when it is at least 60%, more generally 75% or over90%, by weight, of the total material in a sample. Thus, for example, apolypeptide that is chemically synthesised or produced by recombinanttechnology will generally be substantially free from its naturallyassociated components. A nucleic acid molecule is substantially purewhen it is not immediately contiguous with (i.e., covalently linked to)the coding sequences with which it is normally contiguous in thenaturally occurring genome of the organism from which the DNA of theinvention is derived. A substantially pure compound can be obtained, forexample, by extraction from a natural source; by expression of arecombinant nucleic acid molecule encoding a polypeptide compound; or bychemical synthesis. Purity can be measured using any appropriate methodsuch as column chromatography, gel electrophoresis, HPLC, etc.

[0114] “Small molecule” as used herein, is meant to refer to acomposition, which has a molecular weight of less than about 1 kD andmost preferably less than about 0.4 kD. Small molecules can benucleotides, amino acids, peptides, peptidomimetics, carbohydrates,lipids or other organic (carbon containing) molecules. Manypharmaceutical companies have extensive libraries of chemical and/orbiological mixtures, often fungal, bacterial, or algal extracts, whichcan be screened with any of the assays of the invention to identifycompounds that modulate an R-14 bioactivity.

[0115] The term “alkyl” refers to the radical of saturated aliphaticgroups, including straight chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. Typical alkyl groupsinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, etc. The alkylgroups can be (C₁-C₆) alkyl, or, (C₁-C₃) alkyl. A “substituted alkyl”has substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, carbonyl (such as carboxyl, ketones (includingalkylcarbonyl and arylcarbonyl groups), and esters (includingalkyloxycarbonyl and aryloxycarbonyl groups)), thiocarbonyl, acyloxy,alkoxyl, phosphoryl, phosphonate, phosphinate, amino, acylamino, amido,amidine, imino, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate,sulfonate, sulfamoyl, sulfonamido, heterocyclyl, aralkyl, or an aromaticor heteroaromatic moiety. The moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate. For instance, thesubstituents of a substituted alkyl may include substituted andunsubstituted forms of aminos, azidos, iminos, amidos, phosphoryls(including phosphonates and phosphinates), sulfonyls (includingsulfates, sulfonamidos, sulfamoyls and sulfonates), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF₃, —CN and the like. Exemplary substitutedalkyls are described below. Cycloalkyls can be further substituted withalkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substitutedalkyls, —CF₃, —CN, and the like.

[0116] The terms “alkenyl” and “alkynyl” refer to unsaturated aliphaticgroups analogous in length and possible substitution to the alkylsdescribed above, but that contain at least one double or triple bondrespectively. An “alkenyl” is an unsaturated branched, straight chain,or cyclic hydrocarbon radical with at least one carbon-carbon doublebond. The radical can be in either the cis or trans conformation aboutthe double bond(s). Typical alkenyl groups include, but are not limitedto, ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, tert-butenyl,pentenyl, hexenyl, etc. An “alkynyl” is an unsaturated branched,straight chain, or cyclic hydrocarbon radical with at least onecarbon-carbon triple bond. Typical alkynyl groups include, but are notlimited to, ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl,etc.

[0117] A “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result, such as reduction of intraocular pressureand related disorders such as glaucoma. A therapeutically effectiveamount of a compound of the invention may vary according to factors suchas the disease state, age, sex, and weight of the individual, and theability of the compound to elicit a desired response in the individual.Dosage regimens may be adjusted to provide the optimum therapeuticresponse. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the compound are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result, such aspreventing or reducing elevated intraocular pressure and in turnpreventing or treating related disorders such as glaucoma. Aprophylactically effective amount can be determined as described abovefor the therapeutically effective amount. For any particular subject,specific dosage regimens may be adjusted over time according to theindividual need and the professional judgement of the personadministering or supervising the administration of the compositions.

[0118] As used herein “pharmaceutically acceptable carrier” or“excipient” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. In oneembodiment, the carrier is suitable for parenteral administration.Alternatively, the carrier can be suitable for intravenous,intraperitoneal, intramuscular, sublingual or oral administration.Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

[0119] R14 Nucleic Acids

[0120] As noted above, the present invention is based, in part, on thediscovery of a human gene, which encodes a human nucleic acid andencoding a protein referred to as “R-14”. In silico analysis showed thatR-14 could be an integral membrane protein containing seven prominenthydrophobic domains and many signature motifs of a G protein-coupledreceptor. The protein and nucleic acid of R-14 show strong homology tohuman, mouse and rat MAS-related G protein-coupled receptors by BLASTNand BLASTP analyses. A10.96 kb subclone of BAC RP11-206c1 (obtained fromSanger Center, UK) was completely sequenced and determined to containthe entire coding region of R-14 (SEQ ID NO:1). The human R-14 codingsequence is 0.966 kb in size and contains no introns. The 966 bp openreading frame (SEQ ID NO:1) encodes a 322 amino acid polypeptide (SEQ IDNO:2). BLASTN analysis of dBEST data base revealed near identity to theEST termed as HTMI-0025F1 (GenBank Acc.: BE439409) and the nucleic acidsequence is identical to HTMI-0025 (Gonzalez, P. et al., supra) with theexception of two nucleotide changes, A to C leading to Gln to Pro at661158 (NT_(—)009307.3 (Hs11_(—)9464).

[0121] The invention provides an isolated R-14 nucleic acid, homologsthereof, and portions thereof. Preferred nucleic acids have a sequence,which is at least about 60%, 65%, 70%, 75%, 80%, 85% and preferably 90%and 95% homologous with a nucleotide sequence of an R-14 gene, e.g.,such as a sequence shown in SEQ ID NO. 1. Preferred nucleic acids arevertebrate R-14 nucleic acids. Particularly preferred vertebrate R-14nucleic acids are mammalian, in an embodiment, human. In one embodiment,the preferred nucleic acid is a cDNA encoding a polypeptide having atleast one bioactivity of the subject R-14 polypeptide.

[0122] R14 Polypeptides

[0123] The present invention features R-14 polypeptides which can beproduced in and isolated from, cells or tissues in which the thepolypeptide is naturally expressed, or cells in which R-14 polypeptideis expressed using gene transfer of recombinant R1-4 nucleic acids,either as cell-free extracts or purified membrane fractions. Functionalforms of the subject polypeptides can be prepared as purifiedpreparations by using a cloned gene as described herein. Preferred R-14proteins of the invention have an amino acid sequence which is at leastabout 60%, 65%, 70%, 75, 80%, 85%, 90%, or 95% identical or homologousto the amino acid sequence of SEQ ID NO:2. In a preferred embodiment, anR-14 protein of the present invention is a mammalian R-14 protein. In aparticularly preferred embodiment an R-14 protein is set forth as SEQ IDNO:2. It will be understood that certain post-translationalmodifications, e.g., phosphorylation and the like, can increase theapparent molecular weight of the R-14 protein relative to the unmodifiedpolypeptide chain. Protein isoforms encoded by splice variants of R-14listed in SEQ ID NO:2 are also within the scope of the presentinvention. Such isoforms may have additional biological activities fromthose possessed by the R-14 proteins specified by SEQ ID NO:2.

[0124] R14 Polypeptide Fusions, Mutant-Proteins and Homologs Thereof

[0125] In one aspect, the invention relates to R-14 polypeptides, eitheras enriched fractions of cells and tissues, or substantially purepreparations of naturally-occurring or recombinantly-produced orchemically-synthesized polypeptides. An R-14 polypeptide of theinvention can comprise a full length protein as set forth in SEQ ID NO:2or can comprises fusion proteins containing smaller fragmentscorresponding to one or more particular motifs/domains, or fragmentscomprising at least about 100, 125, 150, 175, 200, 225, 250, 275, 300amino acids in length. The subject R-14 protein also includes within itsscope modified proteins, e.g. proteins in which specific mutationsprevent post-translational modification, such as glycosylation,myristylation, palmitylation and phosphorylation of the protein, orwhich mutations prevent or enhance interaction of the R-14 mutantprotein with agonists, antagonists or intracellular proteins involved insignal transduction.

[0126] In further embodiments, R-14 polypeptides of the invention maycomprise a fragment of the polypeptide of SEQ ID NO:2, as defined by aminimum number of consecutive amino acids thereof. Accordingly, inembodiments the invention relates to an R-14 polypeptide comprising atleast 100, 125, 150, 175, 200, 225, 250, 275, 278, 280, 290, 300, 305,310, 315, 320 consecutive amino acids of SEQ ID NO:2.

[0127] In another aspect, the, invention relates to a recombinantexpression system for producing an R-14 protein. For expression incells, tissues and animals, the nucleic acid as set in SEQ. ID NO. 1 wascloned into a mammalian expression vector, in which R-14 nucleic acidwas operably linked to a transcriptional regulatory sequence, e.g., atleast one of a transcriptional promoter (for constitutive expression),sequences required for splicing and transcription termination. Suchregulatory sequences in conjunction with a R-14 nucleic acid moleculeprovided a useful vector for gene expression. People skilled in the artcould use similar strategies to express R-14 protein in prokaryotic andeukaryotic host cells transfected with appropriate expression vectors invitro (e.g. cell culture) and in vivo (e.g. transgenic) methods forproducing R-14 proteins.

[0128] The present invention further pertains to methods of producingthe subject R-14 polypeptides. For example, a “host cell” transfectedwith a nucleic acid “vector” directing expression of a nucleotidesequence encoding the subject polypeptides can be cultured underappropriate conditions to allow expression of the peptide to occur.Suitable media for cell culture are well known in the art. Therecombinant R-14 polypeptide can be isolated from cell culture medium,host cells, or both using techniques known in the art for purifyingproteins including ion-exchange chromatography, gel filtrationchromatography, ultrafiltration, electrophoresis, and immunoaffinitypurification with antibodies specific for such peptides. In a preferredembodiment, the recombinant R-14 polypeptide is a fusion proteincontaining a domain which facilitates its purification, such aspolyhistidine fusion of R-14 protein.

[0129] The invention also provides fusion proteins, e.g., R-14conjugated to green fluorescent protein or beta arrestin. Such fusionproteins can provide detection of R-14 polypeptides in cells, tissuesand organisms. Fusions of green fluorescent protein (GFP) to R-14protein can be used to locate and follow the dynamics of R-14, such asaggregation, association with other cellular proteins, internalization,trafficking, degradation in endocytotic vesicles, in living or fixedcells. R-14 fusions of GFP and luciferase can be used to study andmonitor dimer and oligomer formation, association with other signallingmolecules. R-14-G_(α) protein fusions can be used to measure GTP bindingand hydrolysis by the G protein in response to agonists or antagonistsand these methods, known to people skilled in the art, are used toscreen and/or test small molecule compound libraries for agonist orantagonist activity. These examples are presented to illustrate, but notto limit the potential fusion partners and their uses in basic andapplied scientific studies.

[0130] Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide homologs of one of thesubject R-14 polypeptides, which function in a limited capacity as oneof either an R-14 agonist (mimetic) or an R-14 antagonist, in order topromote or inhibit only a subset of the biological activities of thenaturally-occurring form of the protein. Homologs of each of the subjectR-14 proteins can be generated by mutagenesis, such as by discrete pointmutation(s), or by truncation. For instance, mutation can give rise tohomologs which display elevated ligand-independent activity orsubstantially the same, or merely a subset of the biological activity ofthe R-14 polypeptide from which it was derived. Alternatively,antagonistic forms of the protein can be generated which are able toinhibit the function of the naturally occurring form of the protein,such as by competitively binding to an R-14 receptor.

[0131] R14 Activity and Assaying Said Activity

[0132] In one aspect, the invention provides methods for identifying acompound that can modulate R-14 “activity”. Such a method may entaildetermining the activity of an R-14 protein in the presence of a test orcandidate compound. Such a method may for, example be used to identifyan R-14 antagonist, which may be useful for lowering intraocularpressure, and further for treating a condition associated with elevatedintraocular pressure, such as glaucoma and related conditions. Variousaspects of R-14 activity may be assayed in this regard, as noted hereinand in the Examples.

[0133] In an embodiment, determining R-14 “activity” entails assaying aninteraction between an R-14 polypeptide and an R-14 binding partner, toidentify compounds that are capable of interfering with the interactionof R-14 and its binding partner, and thus the test compound may becapable of binding to an R-14 polypeptide. In an embodiment, such amethod includes the steps of (a) forming a mixture, which includes: (i)an R-14 polypeptide, (ii) an R-14 binding partner and (iii) a testcompound; and (b) detecting interaction of the R-14 polypeptide and theR-14 binding partner or alteration in at least one aspect of R-14polypeptide “activity”. A statistically significant change (potentiationor inhibition) in R-14 activity in the presence of the test compound,relative to that in the absence of the test compound, indicates apotential agonist or antagonist (inhibitor) respectively of R-14bioactivity for the test compound. The reaction mixture can be acell-free protein preparation, e.g., a reconstituted protein mixture ora cell lysate or purified cell constituents, or a cultured cellrecombinantly expressing the R-14 polypeptide or fragments thereof.People skilled in the art can use such a competitive binding assay todetect the interaction between an R-14 polypeptide and a, R-14 bindingpartner. In an embodiment, at least one of the R-14 polypeptide and theR-14 binding partner comprises a detectable label, and interaction ofthe R-14 and R-14 binding partner is quantified by detecting the labelin the complex. The detectable label can be, e.g., a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor.

[0134] Cell-Free Assays

[0135] Cell-free assays can be used to identify compounds which arecapable of interacting with an R-14 protein, thereby modify the activityof the R-14 protein. Such a compound can, e.g., modify the structure of,an R-14 protein and thereby affect its activity. Cell-free assays canalso be used to identify compounds which modulate the interactionbetween an R-14 protein and a R-14 binding partner. In a preferredembodiment, cell-free assays for identifying such compounds consistessentially in a reaction mixture containing an R-14 protein, R-14binding partner and a test compound. A test compound can be, e.g., aderivative of a R-14 polypeptide or R-14 binding partner.

[0136] Accordingly, one exemplary screening assay of the presentinvention includes the steps of (a) forming a reaction mixtureincluding: (i) an R-14 polypeptide, (ii) an R-14 binding partner, and(iii) a test compound; and (b) detecting interaction of the R-14 and theR-14 binding protein. For detection purposes, the binding partner can belabelled with a specific marker such as a radionuclide, or a fluorescentcompound or an enzyme. Interaction of a test compound with an R-14protein or fragment thereof can then be detected by determining thelevel of the marker label after an incubation step and a washing step.The R-14 polypeptide and R-14 binding partner can be producedrecombinantly, purified from a source, e.g., plasma, or chemicallysynthesized, as described herein. A statistically significant change(potentiation or inhibition) in the interaction of the R-14 and R-14binding protein in the presence of the test compound, relative to theinteraction in the absence of the test compound, indicates a potentialagonist (mimetic or potentiator) or antagonist (inhibitor) of R-14bioactivity for the test compound. Radiolabelled samples are counted andquantified by scintillation spectrophotometry. Binding ligands can beconjugated to enzymes such as acetylcholine esterase and boundR-14-binding partner can be quantified by enzyme assay.

[0137] Cell-free assays can also be used to identify compounds whichinteract with an R-14 protein and modulate an activity of an R-14protein. Accordingly, in one embodiment, an R-14 protein is contactedwith a test compound and the bioactivity of R-14 is monitored. Thebioactivity of R-14 protein in cell-free assays include, GTP binding,GTP hydrolysis, Dissociation of G_(□) proteins, adenylate cyclaseactivation, phospholipase (A2, beta, gamma and D isoforms) activation,phospholipid hydrolysis, cAMP synthesis etc. and the methods ofmeasuring these changes in the bioactivity of R-14 protein are wellknown to those skilled in the art.

[0138] Cell Based Assays

[0139] In addition to cell-free assays, such as described above, R-14proteins as provided by the present invention, facilitate the generationof cell-based assays, e.g., for identifying an agent capable ofmodulating R-14 activity, such as small molecule agonists orantagonists. Cell based assays can be used, for example, to identifycompounds which modulate the bioactivity of R-14 protein, expression ofan R-14 gene or those genes that are induced or suppressed in responseto increased or decreased bioactivity of R-14 protein. Accordingly, inone embodiment, a cell which is capable of producing R-14 is incubatedwith a test compound in the presence or absence of a natural orsynthetic agonist/antagonist of R-14 and the bioactivity of R-14 ismeasured. The resultant alterations in the bioactivity of R-14 arecompared to control R-14 producing cells which have not been contactedwith the test compound. These measurements are used to assess thepotency, affinity, action of the test compound towards modulating R-14bioactivity.

[0140] A particular embodiment of the present invention is thatcell-free and cell-based assays involving the use of R-14 protein as setforth in SEQ ID NO. 2, could become an integral part of a screeningsystem to evaluate and select small molecules which can be optimized tobe used as therapeutics for lowering intraocular pressure and in thetreatment of glaucoma.

[0141] Kits

[0142] The invention further provides kits for use in diagnostics orscreening methods for providing R-14 binding partners or glaucomatherapeutics. For example, the kit can comprise (1) a labeled R-14binding partner and/or (2) cell-free lysates or cellular fractionsincluding membranes isolated from R-14 expressing host cells orR-14-expressing tissues or whole cells expressing R-14 protein naturallyor by recombinant DNA methods and/or (3) an agent capable of detectingR-14 protein or mRNA and/or (4) means for determining the amount of R-14protein, mRNA or bioactivity and the means for comparing the amount ofR-14 protein, mRNA or bioactivity in the sample with a standard. Thecompound or agent can be packaged in a suitable container. The kit canfurther comprise instructions for using the kit to detect R-14 mRNA orprotein or its bioactivity. Such a kit can comprise, e.g., one or morenucleic acid probes capable of hybridizing specifically to at least aportion of an R-14 gene or allelic variant thereof, or mutated formthereof.

[0143] R14 as a Drug Target in Intraocular Hypertension

[0144] Applicants' results described herein indicate that R-14 proteinrepresents a drug target, i.e. with a view to provide R-14 modulatorswhich may be useful for lowering intraocular pressure and thus may beuseful for the treatment of conditions associated with elevatedintraocular pressure, such as glaucoma and related conditions. In a morepreferred embodiment, the invention provides a method by which R-14 geneand protein can be expressed in host cells such as mammalian cells, andthe cells be used for small molecule or peptide lead compound discoveryin order to provide pharmaceutical compositions useful in treatingpatients diagnosed with elevated intraocular pressure and relatedconditions such as glaucoma.

[0145] R14 Antagonists, Pharmaceutical Compositions, Uses Thereof

[0146] Applicants have further identified and characterized agents (e.g.peptide compounds) capable of modulating R-14 activity, e.g. can act aspeptide antagonists of R-14 activity. Accordingly, the inventionprovides peptide compounds for use in lowering intraocular pressure. Ina further embodiment, the invention provides peptide compounds for usein the treatment of a condition associated with elevated intraocularpressure such as glaucoma and related conditions. The invention furtherprovides a method of lowering intraocular pressure in a subject and amethod for the treatment of a condition associated with elevatedintraocular pressure such as glaucoma and related conditions, themethods comprising administering an effective amount of apeptide/peptide compound of the invention, or a composition comprising apeptide of the invention, to the subject, e.g. a subject in needthereof. In an embodiment, the subject is a mammal, in a furtherembodiment, a human.

[0147] Therefore, in an aspect, the invention provides a substantiallypure peptide compound of Formula I:

Z₁-X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈-Z₂  I

[0148] wherein:

[0149] X₁ is selected from the group consisting of Phe, His, Ile andrelated amino acids;

[0150] X₂ is selected from the group consisting of Ser, Ile, Phe andrelated amino acids

[0151] X₃ is selected from the group consisting of Leu, Ile, Asp andrelated amino acids

[0152] X₄ is selected from the group consisting of Thr, Cys, Ser andrelated amino acids possessing side chains containing sulfhydryl,hydroxyl or H-bond forming groups;

[0153] X₅ is selected from the group consisting of Gln, Ser, Thr andrelated amino acids;

[0154] X₆ is selected from the group consisting of Lys, Pro, Glu andrelated amino acids;

[0155] X₇ is selected from the group consisting of Tyr, Leu, Cys andrelated amino acids;

[0156] X₈ is selected from the group consisting of Cys, Arg, Trp andrelated amino acids;

[0157] Z₁ is an N-terminal group of the formula H₂N—, RHN— or, RRN—;

[0158] Z₂ is a C-terminal group of the formula —C(O)OH, —C(O)R, —C(O)OR,—C(O)NHR, —C(O)NRR;

[0159] R at each occurrence is independently selected from (C₁-C₆)alkyl, (C₁-C₆) alkenyl, (C₁-C₆) alkynyl, substituted (C₁-C₆) alkyl,substituted (C₁-C₆) alkenyl, or substituted (C₁-C₆) alkynyl; and

[0160] “-” is a covalent linkage.

[0161] In a further embodiment, Z₁ is an N-terminal group selected fromthe group consisting of a proton, a sequence of 1-3 amino acids, or ablocking group such as a carbamate group, an acyl group composed of ahydrophobic moiety such as cyclohexyl, phenyl, benzyl, short chainlinear and branched alkyl groups of 1-8 carbons. In a furtherembodiment, Z₂ is a carboxy-terminal group selected from the groupconsisting of a proton, NH₂, 1-3 amino acids as well as arylalkyl aminessuch as benzylamine, phenylethylamine, phenylpropylamine, and aliphaticamines possessing short chain linear and branched alkyl groups of 1 to 8carbons.

[0162] The invention further provides a substantially pure syntheticpeptide compound or recombinant peptide compound, said compound having adomain of Formula II:

—X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈—  II

[0163] wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈ and “-” are defined asabove.

[0164] In embodiments, the peptide compound comprises a sequenceselected from the group consisting of:

[0165] NH₂-Phe Ser Leu Thr Gln Lys Tyr Cys-OH (FSLTQKYC; SEQ ID NO:3);

[0166] NH₂-His Ile Ile Cys Ser Pro Leu Arg-OH (HIICSPLR; SEQ ID NO:4);and

[0167] NH₂-Ile Phe Asp Ser Thr Gln-Cys Trp-OH (IFDSTECW; SEQ ID NO:5).

[0168] The invention further provides an active fragment of the abovenoted peptides. “Active fragment” refers to a fragment of a peptide ofthe invention which is capable of modulating the activity of an R-14protein, wherein said R-14 protein comprises a polypeptide selected fromthe group consisting of:

[0169] (a) the polypeptide of SEQ ID NO. 2; and

[0170] (b) a polypeptide encoded by a first nucleic acid that issubstantially identical to a second nucleic acid capable of encoding thepolypeptide of SEQ ID NO. 2.

[0171] The invention further provides derivatives of the above (SEQ IDNOs:3, 4 and 5) which may be synthetic polypeptides containingconservative substitutions of individual amino acids, andpeptidomimetics thereof.

[0172] The invention further provides a recombinant expression system,vectors and host cells, such as those described above, for theexpression/production of a peptide comprising a peptide of the invention(such as those set forth in SEQ ID NOs. 3, 4 and 5), using for exampleculture media, production, isolation and purification methods such asthose described above. Such vectors comprise a nucleic acid sequencecapable of encoding such a peptide operably linked to a transcriptionalregulatory sequence. In an embodiment, the peptide is a fusion peptidecontaining a domain which facilitates its purification, such as apolyhistidine domain.

[0173] The invention also aims to provide a pharmaceutical compositioncomprising a peptidic or peptidomimetic compound with a pharmaceuticallyacceptable carrier, wherein said compound is capable of modulating, inan embodiment inhibiting at least one aspect of R-14 polypeptidebioactivity.

[0174] The invention also aims to provide a pharmaceutical compositioncontaining a peptidic or peptidomimetic compound with a pharmaceuticallyacceptable carrier, wherein said compound is capable of decreasingintraocular pressure.

[0175] Also within the scope of the invention are pharmaceuticalcompositions for treating patients diagnosed with increased intraocularpressure and/or glaucoma, comprising administering (e.g., either locallyor systemically) to a subject, a pharmaceutically effective amount of acomposition comprising a compound capable of modulating at least oneaspect of R-14 bioactivity.

[0176] R14 Antagonists, Active Fragments, Peptidomimetics Thereof

[0177] The invention also provides for reduction of the fragments ofR-14 antagonists to generate mimetics, e.g., peptide or non-peptideagents, such as small molecules, which are agonistic or antagonistic ofR-14 protein activity.

[0178] In order to improve the R-14 antagonists described in thisinvention for therapeutic use, several modifications of the peptide canbe made by substituting a first amino acid with a “related amino acid”which is a second amino acid related to the first amino acid by eitherstructure or function of the side chain: aromatic, aliphatic,positively- or negatively-charged. Examples of related amino acids areprovided in Tables 2 and 3 below. TABLE 2 Examples of related aminoacids Residue Substitution Ala Gly; Ser Arg Lys Asn Gln; His Asp Glu CysSer Gln Asn Glu Asp Gly Ala; Pro His Asn; Gln Ile Leu; Val Leu Ile; ValLys Arg Met Leu; Ile; Val Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr;His; Phe Tyr Trp; Phe Val Ile; Leu Pro Ala; Gly

[0179] Alternatively, another group of substitutions of the R14antagonists of the present invention are those in which at least oneamino acid residue has been removed and a different residue inserted inits place according to the following Table 2. Another group ofsubstitutions are defined herein as exchanges within one of thefollowing five groups: TABLE 3 Relations among amino acids Smallaliphatic, nonpolar or Ala, Ser, Thr, slightly polar residues (Pro, Gly)Polar, negatively charged Asp, Asn, Glu, residues and their amides GlnPolar, positively charged His, Arg, Lys residues Large aliphatic,nonpolar Met, Leu, Ile, residues Val, (Cys) Aromatic residues Phe, Tyr,Trp

[0180] The three amino acid residues in parentheses above have specialroles in protein architecture. Gly is the only residue lacking any sidechain and thus imparts flexibility to the chain. This however tends topromote the formation of secondary structure other than alpha-helical.Pro, because of its unusual geometry, tightly constrains the chain. Itgenerally tends to promote beta turn-like structures. Cys is capable ofparticipating in disulfide bond formation. Tyr, because of its hydrogenbonding potential, has significant kinship with Ser, and Thr, etc.

[0181] In addition, any amino acid representing a component of the saidpeptides can be replaced by the same amino acid but of the oppositechirality. Thus, any amino acid naturally occurring in theL-configuration (which may also be referred to as the R or S, dependingupon the structure of the chemical entity) may be replaced with an aminoacid of the same chemical structural type, but of the oppositechirality, generally referred to as the D-amino acid but which canadditionally be referred to as the R- or the S-, depending upon itscomposition and chemical configuration. Additional variations include b-and g-amino acids that provide different spatial arrangement of chemicalgroups.

[0182] In addition to the substitutions outlined above, synthetic aminoacids that provide similar side chain functionality can be introduced into the peptide. For example, aromatic amino acids may be replaced withD- or L-naphthylalanine, D- or L-Phenylglycine, D- orL-2-thienylalanine, D- or L-1-, 2-, 3- or 4-pyrenylalanine, D- orL-3-thienylalanine, D- or L-(2-pyridinyl)-alanine, D- orL-(3-pyridinyl)-alanine, D- or L-(2-pyrazinyl)-alanine, D- orL-(4-isopropyl)-phenylglycine, D-(trifluoromethyl)-phenylglycine,D-(trifluoromethyl)-phenylalanine, D-p-fluorophenylalanine, D- orL-p-biphenylalanine D- or L-p-methoxybiphenylalanine, D- orL-2-indole(alkyl)alanines, and D- or L-alkylalanines where alkyl may besubstituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl,pentyl, isopropyl, iso-butyl, iso-pentyl groups. Non-carboxylate aminoacids can be made to possess negative charge, such as the non-limitingexamples of phosphono- or sulfated (e.g. —SO₃H) amino acids.

[0183] Other substitutions may include unnatural alkylated amino acidswhich are made by combining an alkyl group with any natural amino acid.Basic natural amino acids such as lysine, arginine may be substitutedwith alkyl groups at NH₂. Others are nitrile derivatives (e.g.,containing the CN-moiety in place of CONH₂) of asparagine or glutamine,and sulfoxide derivative of methionine. In addition, any amide linkagein the peptide can be replaced by a ketomethylene, hydroxyethyl,ethyl/reduced amide, thioamide or reversed amide moieties, e.g.(—C═O)—CH₂—), (—CHOH)—CH₂—), (CH₂—CH₂—), (—C═S)—NH—), or (—NH—(—C═O) for(—C═O)—NH—).

[0184] Compounds of the invention can be prepared, for example, byreplacing, deleting, or inserting an amino acid residue of a peptidecompound or domain of the invention, with other conservative amino acidresidues, i.e., residues having similar physical, biological, orchemical properties, and screening for biological function. It is wellknown in the art that some modifications and changes can be made in thestructure of a polypeptide without substantially altering the biologicalfunction of that peptide, to obtain a biologically equivalentpolypeptide. The peptides, ligands and domains of the present inventionalso extend to biologically equivalent peptides, ligands and domainsthat differ from a portion of the sequence of novel ligands of thepresent invention by conservative amino acid substitutions. As usedherein, the term “conserved amino acid substitutions” refers to thesubstitution of one amino acid for another at a given location in thepeptide, where the substitution can be made without substantial loss ofthe relevant function. In making such changes, substitutions of likeamino acid residues can be made on the basis of relative similarity ofside-chain substituents, for example, their size, charge,hydrophobicity, hydrophilicity, and the like, and such substitutions maybe assayed for their effect on the function of the peptide by routinetesting.

[0185] In some embodiments, conserved amino acid substitutions may bemade where an amino acid residue is substituted for another having asimilar hydrophilicity value (e.g., within a value of plus or minus2.0), where the following may be an amino acid having a hydropathicindex of about −1.6 such as Tyr (−1.3) or Pro (−1.6)s are assigned toamino acid residues (as detailed in U.S. Pat. No. 4,554,101,incorporated herein by reference): Arg (+3.0); Lys (+3.0); Asp (+3.0);Glu (+3.0); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Pro (−0.5); Thr(−0.4); Ala (−0.5); His (−0.5); Cys (−1.0); Met (−1.3); Val (−1.5); Leu(−1.8); Ile (−1.8); Tyr (−2.3); Phe (−2.5); and Trp (−3.4).

[0186] In alternative embodiments, conserved amino acid substitutionsmay be made where an amino acid residue is substituted for anotherhaving a similar hydropathic index (e.g., within a value of plus orminus 2.0). In such embodiments, each amino acid residue may be assigneda hydropathic index on the basis of its hydrophobicity and chargecharacteristics, as follows: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe(+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (−0.4); Thr (−0.7); Ser(−0.8); Trp (−0.9); Tyr (−1.3); Pro (−1.6); His (−3.2); Glu (−3.5); Gln(−3.5); Asp (−3.5); Asn (−3.5); Lys (−3.9); and Arg (−4.5).

[0187] In alternative embodiments, conserved amino acid substitutionsmay be made where an amino acid residue is substituted for another inthe same class, where the amino acids are divided into non-polar,acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu,Ile, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His;neutral: Gly, Ser, Thr, Cys, Asn, Gln, Tyr.

[0188] Conservative amino acid changes can include the substitution ofan L-amino acid by the corresponding D-amino acid, by a conservativeD-amino acid, or by a naturally-occurring, non-genetically encoded formof amino acid, as well as a conservative substitution of an L-aminoacid. Naturally-occurring non-genetically encoded amino acids includebeta-alanine, 3-amino-propionic acid, 2,3-diamino propionic acid,alpha-aminoisobutyric acid, 4-amino-butyric acid, N-methylglycine(sarcosine), hydroxyproline, ornithine, citrulline, t-butylalanine,t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine,norleucine, norvaline, 2-napthylalanine, pyridylalanine, 3-benzothienylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine,3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine,1,2,3,4-tetrahydro-isoquinoline-3-carboxylix acid,beta-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyllysine, 2-amino butyric acid, 2-amino butyric acid, 2,4,-diamino butyricacid, p-aminophenylalanine, N-methylvaline, homocysteine, homoserine,cysteic acid, epsilon-amino hexanoic acid, delta-amino valeric acid, or2,3-diaminobutyric acid.

[0189] In alternative embodiments, conservative amino acid changesinclude changes based on considerations of hydrophilicity orhydrophobicity, size or volume, or charge. Amino acids can be generallycharacterized as hydrophobic or hydrophilic, depending primarily on theproperties of the amino acid side chain. A hydrophobic amino acidexhibits a hydrophobicity of greater than zero, and a hydrophilic aminoacid exhibits a hydrophilicity of less than zero, based on thenormalized consensus hydrophobicity scale of Eisenberg et al. (J. Mol.Bio. 179:125-142, 1984). Genetically encoded hydrophobic amino acidsinclude Gly, Ala, Phe, Val, Leu, Ile, Pro, Met and Trp, and genetically,encoded hydrophilic amino acids include Thr, His, Glu, Gln, Asp, Arg,Ser, and Lys. Non-genetically encoded hydrophobic amino acids includet-butylalanine, while non-genetically encoded hydrophilic amino acidsinclude citrulline and homocysteine.

[0190] Hydrophobic or hydrophilic amino acids can be further subdividedbased on the characteristics of their side chains. For example, anaromatic amino acid is a hydrophobic amino acid with a side chaincontaining at least one aromatic or heteroaromatic ring, which maycontain one or more substituents such as —OH, —SH, —CN, —F, —Cl, —Br,—I, —NO₂, —NO, —NH₂, —NHR, —NRR, —C(O)R, —C(O)OH, —C(O)OR, —C(O)NH₂,—C(O)NHR, —C(O)NRR; etc., where R is independently (C₁-C₆) alkyl,substituted (C₁-C₆) alkyl, (C₁-C₆) alkenyl, substituted (C₁-C₆) alkenyl,(C₁-C₆) alkynyl, substituted (C₁-C₆) alkynyl, (C₅-C₂₀) aryl, substituted(C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 memberedalkheteroaryl or substituted 6-26 membered alkheteroaryl. Geneticallyencoded aromatic amino acids include Phe, Tyr, and Tryp, whilenon-genetically encoded aromatic amino acids include phenylglycine,2-napthylalanine, beta-2-thienylalanine,1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid,4-chlorophenylalanine, 2-fluorophenylalanine3-fluorophenylalanine, and4-fluorophenylalanine.

[0191] An apolar amino acid is a hydrophobic amino acid with a sidechain that is uncharged at physiological pH and which has bonds in whicha pair of electrons shared in common by two atoms is generally held,equally by each of the two atoms (i.e., the side chain is not polar).Genetically encoded apolar amino acids include Gly, Leu, Val, Ile, Ala,and Met, while non-genetically encoded apolar amino acids includecyclohexylalanine. Apolar amino acids can be further subdivided toinclude aliphatic amino acids, which is a hydrophobic amino acid havingan aliphatic hydrocarbon side chain. Genetically encoded aliphatic aminoacids include Ala, Leu, Val, and Ile, while non-genetically encodedaliphatic amino acids include norleucine.

[0192] A polar amino acid is a hydrophilic amino acid with a side chainthat is uncharged at physiological pH, but which has one bond in whichthe pair of electrons shared in common by two atoms is held more closelyby one of the atoms. Genetically encoded polar amino acids include Ser,Thr, Asn, and Gln, while non-genetically encoded polar amino acidsinclude citrulline, N-acetyl lysine, and methionine sulfoxide.

[0193] An acidic amino acid is a hydrophilic amino acid with a sidechain pKa value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Genetically encoded acidic amino acids include Asp andGlu. A basic amino acid is a hydrophilic amino acid with a side chainpKa value of greater than 7. Basic amino acids typically have positivelycharged side chains at physiological pH due to association withhydronium ion. Genetically encoded basic amino acids include Arg, Lys,and His, while non-genetically encoded basic amino acids include thenon-cyclic amino acids ornithine, 2,3,-diaminopropionic acid,2,4-diaminobutyric acid, and homoarginine.

[0194] The above classifications are not absolute and an amino acid maybe classified in more than one category. In addition, amino acids can beclassified based on known behaviour and or characteristic chemical,physical, or biological properties based on specified assays or ascompared with previously identified amino acids. Amino acids can alsoinclude bifunctional moieties having amino acid-like side chains.

[0195] Conservative changes can also include the substitution of achemically derivatised moiety for a non-derivatised residue, by forexample, reaction of a functional side group of an amino acid. Thus,these substitutions can include compounds whose free amino groups havebeen derivatised to amine hydrochlorides, p-toluene sulfonyl groups,carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups orformyl groups. Similarly, free carboxyl groups can be derivatized toform salts, methyl and ethyl esters or other types of esters orhydrazides, and side chains can be derivatized to form O-acyl or O-alkylderivatives for, free hydroxyl groups or N-im-benzylhistidine for theimidazole nitrogen of histidine. Peptide analogues also include aminoacids that have been chemically altered, for example, by methylation, byamidation of the C-terminal amino acid by an alkylamine such asethylamine, ethanolamine, or ethylene diamine, or acylation ormethylation of an amino acid side chain (such as acylation of theepsilon amino group of lysine). Peptide analogues can also includereplacement of the amide linkage in the peptide with a substituted amide(for example, groups of the formula —C(O)—NR, where R is (C₁-C₆) alkyl,(C₁-C₆) alkenyl, (C₁-C₆) alkynyl, substituted (C₁-C₆) alkyl, substituted(C₁-C₆) alkenyl, or substituted (C₁-C₆) alkynyl) or isostere of an amidelinkage (for example, —CH₂NH—, —CH₂S, —CH₂CH₂—, —CH═CH— (cis and trans),—C(O)CH₂—, —CH(OH)CH₂—, or —CH₂SO—).

[0196] In order to improve the pharmaceutical characteristics of theR-14 antagonists, the size of the peptides can be reduced by deletingone or more amino acids and use amino acid mimetics or dipeptide mimicscontaining non-peptide bonds. Examples of using molecular scaffolds suchas benzodiazepine, azepine, substituted gamma lactam rings,keto-methylene pseudopeptides, β-turn dipeptide cores andβ-aminoalcohols for these purposes are known to peptide chemists and aredescribed in in Peptidomimetic protocols (Methods in molecular medicineVol. 23) W. M. Kazmierski (ed.), Humana Press and Advances in Amino AcidMimetics and Peptidomimetics, Vols. 1 & 2 A. Abell (Ed).

[0197] Covalent modifications of the peptide are thus included withinthe scope of the present invention. Such modifications may be introducedinto the R-14 antagonists by reacting targeted amino acid residues ofthe polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues. The followingexamples of chemical derivatives are provided by way of illustration andnot by way of limitation. Cysteinyl residues may be reacted withalpha-haloacetates (and corresponding amines), such as 2-chloroaceticacid or chloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Histidyl residues may be derivatized by reaction withcompounds such as diethylprocarbonate e.g., at pH 5.5-7.0 because thisagent is relatively specific for the histidyl side chain, andpara-bromophenacyl bromide may also be used; e.g., where the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0. Lysinyl andamino terminal residues may be reacted with compounds such as succinicor other carboxylic acid anhydrides. Other suitable reagents forderivatizing alpha-amino-containing residues include compounds such asimidoesters/e.g. as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

[0198] Arginyl residues may be modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin according to known method steps.Derivatization of arginine residues requires that the reaction beperformed in alkaline conditions because of the high pKa of theguanidine functional group. Furthermore, these reagents may react withthe groups of lysine as well as the arginine epsilon-amino group. Thespecific modification of tyrosinyl residues per se is well-known, suchas for introducing spectral labels into tyrosinyl residues by reactionwith aromatic diazonium compounds or tetranitromethane. N-acetylimidazoland tetranitromethane may be used to form O-acetyl tyrosinyl species and3-nitro derivatives, respectively.

[0199] Carboxyl side groups (aspartyl or glutamyl) may be selectivelymodified by reaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermoreaspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions. Glutaminyl andasparaginyl residues may be frequently deamidated to the correspondingglutamyl and aspartyl residues. Other modifications of the peptides inthe present invention may include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the alpha-amino groups of lysine, arginine, and histidineside chains acetylation of the N-terminal amine, methylation of mainchain amide residues (or substitution with N-methyl amino acids) and, insome instances, amidation of the C-terminal carboxyl groups, accordingto known method steps.

[0200] Covalent attachment of fatty acids (C6-C18) to the peptidesconfer additional biological properties such as protease resistance,plasma protein binding, increased plasma half life, intracellularpenetration etc. The above description of modification of a R14antagonist peptides does not limit the scope of the approaches nor thepossible modifications that can be engineered.

[0201] Peptides or peptide analogues can be synthesised by standardchemical techniques, for example, by automated synthesis using solutionor solid phase synthesis methodology. Automated peptide synthesisers arecommercially available and use techniques well known in the art.Peptides and peptide analogues can also be prepared using recombinantDNA technology using standard methods. Accordingly, the inventionfurther provides nucleic acids that encode peptide compounds of theinvention. Such nucleic acids may be introduced into cells forexpression using standard recombinant techniques for stable or transientexpression. Nucleic acid molecules of the invention may include anychain of two or more nucleotides including naturally occurring ornon-naturally occurring nucleotides or nucleotide analogues.

[0202] Methods of Treatment

[0203] The present invention provides for both prophylactic andtherapeutic methods of treating a subject having elevated intraocularpressure and/or glaucoma and related conditions. Therefore, theinvention further provides a method for lowering intraocular pressure orfor treating a condition associated with elevated intraocular pressure,such as glaucoma and related conditions, in a subject, the methodcomprising administration of an agent which is capable of R-14antagonist activity. Administration of a prophylactic agent can occurprior to the manifestation of symptoms characteristic of the R-14aberrancy, such that glaucoma is prevented or, alternatively, itsprogression delayed. In general, the prophylactic or therapeutic methodscomprise administering to the subject an effective amount of a compoundwhich is capable of antagonizing a wildtype R-14 activity or agonizing amutant (defective) R-14 activity. Examples of suitable compounds includethe antagonists, agonists or homologues described in detail herein.

[0204] Effective Dose

[0205] Toxicity and therapeutic efficacy of agents capable of modulatingR-14 activity, such as R-14 agonists or antagonists, can be determinedby standard pharmaceutical procedures in experimental animals, e.g., fordetermining The LD₅₀ (The Dose Lethal To 50% Of The Population) and TheED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds whichexhibit large therapeutic induces are preferred. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. A dose may be formulated in animal models toachieve a circulating plasma concentration range that includes the IC₅₀(i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) determined in in vitro and ex vivoassays and animal studies. Such information can be used to moreaccurately determine useful doses in humans. Levels of R-14 therapeuticsin plasma may be measured, for example, by high performance liquidchromatography. The effective dose of a R-14 therapeutic (agonist orantagonist) could be 0.01 micrograms-100 mg and is determined by theroute of administration, pharmaceutical preparation and the mode ofdelivery.

[0206] Formulation and Use

[0207] Pharmaceutical compositions for use in accordance with thepresent invention may be formulated in conventional manner using one ormore physiologically acceptable carriers or excipients. Thus, thecompounds and their physiologically acceptable salts and solvates may beformulated for administration by, for example, injection, inhalation(either through the mouth or the nose) or oral, buccal, parenteral orrectal administration. Techniques and formulations generally may befound in Reminington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa). For topical administration, R-14 therapeutics of theinvention are formulated into solutions, ointments, salves, gels, orcreams as generally known in the art. For example, a solution containinga R-14 therapeutic can be applied as drops directly on the eye to lowerintraocular pressure.

[0208] In one embodiment, such compositions include an agent capable ofmodulating R-14 activity, such as an R-14 antagonist, in atherapeutically or prophylactically effective amount sufficient toreduce intraocular pressure, and a pharmaceutically acceptable carrier.

[0209] Therapeutic compositions typically must be sterile and stableunder the conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, liposome, or other orderedstructure suitable to high drug concentration. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, monostearate salts and gelatin. Moreover, the R-14 antagonistscan be administered in a time release formulation, for example in acomposition which includes a slow release polymer. The active compoundscan be prepared with carriers that will protect the compound againstrapid release, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylacticacid and polylactic, polyglycolic copolymers (PLG). Many methods for thepreparation of such formulations are patented or generally known tothose skilled in the art.

[0210] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g. R-14 antagonist) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.In accordance with an alternative aspect of the invention, an R-14antagonist may be formulated with one or more additional compounds thatenhance the solubility of the R-14 antagonist.

[0211] A further form of administration is to the eye. An agent orcompound capable of modulating R-14 activity, such as an R-14antagonist, may be delivered in a pharmaceutically acceptable ophthalmicvehicle, such that the compound is maintained in contact with the ocularsurface for a sufficient time period to allow the compound to penetratethe corneal and internal regions of the eye, as for example the anteriorchamber, posterior chamber, vitreous body, aqueous humor, vitreoushumor, cornea, iris/ciliary, lens, choroid/retina and sclera. Thepharmaceutically-acceptable ophthalmic vehicle may, for example, be anointment, vegetable oil or an encapsulating material. Alternatively, thecompounds of the invention may be injected directly into the vitreousand aqueous humour. In a further alternative, the compounds may beadministered systemically, such as by intravenous infusion or injection,for treatment of the eye.

[0212] A further aspect of the present invention is a method of loweringintraocular pressure in a subject and/or preventing and/or treating acondition associated with elevated intraocular pressure (e.g. glaucoma),by administering to a subject a nucleic acid molecule encoding a peptidecompound of the invention. Suitable methods of administration includegene therapy methods.

[0213] A nucleic acid of the invention may be delivered to cells in vivousing methods such as direct injection of DNA, receptor-mediated DNAuptake, viral-mediated transfection or non-viral transfection and lipidbased transfection, all of which may involve the use of gene therapyvectors. Direct injection has been used to introduce naked DNA intocells in vivo (see e.g., Acsadi et al. (1991) Nature 332:815-818; Wolffet al. (1990) Science 247:1465-1468). A delivery apparatus (e.g., a“gene gun”) for injecting DNA into cells in vivo may be used. Such anapparatus may be commercially available (e.g., from BioRad). Naked DNAmay also be introduced into cells by complexing the DNA to a cation,such as polylysine, which is coupled to a ligand for a cell-surfacereceptor (see for example Wu, G. and Wu, C. H. (1988) J. Biol. Chem.263:14621; Wilson el al. (1992) J. Biol. Chem. 267:963-967; and U.S.Pat. No. 5,166,320). Binding of the DNA-ligand complex to the receptormay facilitate uptake of the DNA by receptor-mediated endocytosis. ADNA-ligand complex linked to adenovirus capsids which disrupt endosomes,thereby releasing material into the cytoplasm, may be used to avoiddegradation of the complex by intracellular lysosomes (see for exampleCuriel el al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano etal. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).

[0214] Defective retroviruses, are well characterized for use as genetherapy vectors (for a review see Miller, A. D. (1990) Blood 76:271).Protocols for producing recombinant retroviruses and for infecting cellsin vitro or in vivo with such viruses can be found in Current Protocolsin Molecular Biology, Ausubel, F. M. et al. (eds.) Greene PublishingAssociates, (1989), Sections 9.10-9.14 and other standard laboratorymanuals. Examples of suitable retroviruses include pLJ, pZIP, pWE andpEM which are well known to those skilled in the art. Examples ofsuitable packaging virus lines include .psi.Crip, .psi.Cre, .psi.2 and.psi.Am. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, endothelialcells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitroand/or in vivo (see for example Eglitis, et al. (1985) Science230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

[0215] For use as a gene therapy vector, the genome of an adenovirus maybe manipulated so that it encodes and expresses a peptide compound ofthe invention, but is inactivated in terms of its ability to replicatein a normal lytic viral life cycle. See for example Berkner et al.(1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434;and Rosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 dl324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled inthe art. Recombinant adenoviruses are advantageous in that they do notrequire dividing cells to be effective gene delivery vehicles and can beused to infect a wide variety of cell types, including airway epithelium(Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand etal. (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herzand Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and musclecells (Quantin el al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584).

[0216] Adeno-associated virus (AAV) may be used as a gene therapy vectorfor delivery of DNA for gene therapy purposes. AAV is a naturallyoccurring defective virus that requires another virus, such as anadenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle (Muzyczka et al. Curr. Topics inMicro. and Immunol. (1992) 158:97-129). AAV may be used to integrate DNAinto non-dividing cells (see for example Flotte et al. (1992) Am. J.Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol.63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). AnAAV vector such as that described in Tratschin et al. (1985) Mol. Cell.Biol. 5:3251-3260 may be used to introduce DNA into cells (see forexample Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).Lentiviral gene therapy vectors may also be adapted for use in theinvention.

[0217] General methods for gene therapy are known in the art. See forexample, U.S. Pat. No. 5,399,346 by Anderson et al. A biocompatiblecapsule for delivering genetic material is described in PCT PublicationWO 95/05452 by Baetge et al. Methods of gene transfer into hematopoieticcells have also previously been reported (see Clapp, D. W., et al.,Blood 78: 1132-1139 (1991); Anderson, Science 288:627-9 (2000); andCavazzana-Calvo et al., Science 288:669-72 (2000)).

[0218] The invention further relates to transplantation methods, tointroduce into a subject a cell comprising a nucleic acid capable ofencoding a peptide compound of the invention. The nucleic acid may bepresent in a vector as described above, the vector being introduced intothe cell in vitro, using for example the methods described above. In anembodiment, the cell is autologous, and is obtained from the subject. Inembodiments, the cell is allogeneic or xenogeneic.

[0219] In embodiments, the therapeutic method may be used in conjunctionwith a diagnostic method. For example, a subject suffering from acondition associated with intraocular pressure (e.g. glaucoma) may beidentified or diagnosed using a diagnostic method and then subsequentlytreated using a therapeutic method. Further, the therapeutic method maybe used for treatment in conjunction with the diagnostic or prognosticmethod which is used to monitor the progress of the treatment.

[0220] In accordance with another aspect of the invention, therapeuticcompositions of the present invention, comprising a R-14 antagonist, maybe provided in containers or commercial packages which further compriseinstructions for use of the R-14 antagonist for the prevention and/ortreatment of elevated intraocular pressure and related disorders such asglaucoma.

[0221] Accordingly, the invention further provides a commercial packagecomprising an R-14 antagonist or the above-mentioned compositiontogether with instructions for the prevention and/or treatment ofelevated intraocular pressure and related disorders such as glaucoma.

[0222] The invention further provides a use of the above-noted peptides,compounds and compositions for lowering intraocular pressure in asubject and/or for the prevention and/or treatment of elevatedintraocular pressure and related disorders such as glaucoma.

[0223] The invention further provides a use of the above-noted peptides,compounds and compositions for the preparation of a medicament forlowering intraocular pressure in a subject and/or for the preventionand/or treatment of elevated intraocular pressure and related disorderssuch as glaucoma.

[0224] Although various embodiments of the invention are disclosedherein, many adaptations and modifications may be made within the scopeof the invention in accordance with the common general knowledge ofthose skilled in this art. Such modifications include the substitutionof known equivalents for any aspect of the invention in order to achievethe same result in substantially the same way. Numeric ranges areinclusive of the numbers defining the range. In the claims, the word“comprising” is used as an open-ended term, substantially equivalent tothe phrase “including, but not limited to”. The following examples areillustrative of various aspects of the invention, and do not limit thebroad aspects of the invention as disclosed herein.

EXAMPLES Example 1 Cloning and Characterization of the Nucleic AcidEncoding R-14

[0225] In silico analysis of EST # HTM1-025F1 (GenBank Acc.: BE439409)against human genome using BLAST-N (v. 2.2.1) program resulted in a hitof 99.8% similarity to HTM1-025F1 in contig NT_(—)009307.3, AC020568.4on chromosome 11. A 966 bp fragment was amplified from a BAC clone,RP11-206C1 obtained from the Sanger Center, UK, by PCR, using genespecific primers, 5′ GATTCAACCATCCCAGTCTTGGGTACAG 3′ (SEQ ID NO:6) and5′ TTACTGCTCCAATCTGCTTCCCGACAGC 3′ (SEQ ID NO:7). PCR was done using TaqHiFi (Invitrogen, CA) following the protocol suggested by themanufacturer (annealing at 60° C. for 30 sec, elongation at 68° C. for80 sec: total cycles—35). The PCR products were separated using 1%agarose gels. A photograph of the gel is shown in FIG. 1(A). −Bac: Noplasmid DNA; +Bac: contains plasmid DNA (0.1 μg); MW std: λ Hind IIIdigest. The ˜960 bp fragment (indicated by arrow) was cloned intopcDNA4HisMax TOPO TA (Invitrogen, CA) according to the manufacturer'sinstructions. Orientation of the cloned insert was assessed by PCR usingvector-specific and gene-specific primers (5′ TATGGCTAGCATGACTGGT 3′(SEQ ID NO:8) vector's Express epitope coding sequence and 5′TTACTGCTCCAATCTGCTTCCCGACAGC 3′ (SEQ ID NO:7) gene specific 3′ endprimer). Three clones were sequenced by the dideoxy sequencing method.

[0226] The nucleotide sequence of the R-14 reading frame is shown inFIG. 1(B). The sequences obtained from 2 clones were 99.5% homologous toHTMI-0025 (Gonzalez et al., supra) except for a C (instead of an A) atposition 626. R14.0 protein sequence was 99.4% homologous to MGRx3, agene sequence identified during a search of mas-oncogene relatedsequences in mouse and human genomes (Dong X et al. [2000] Cell.106:619).

[0227] The R-14 polypeptide sequence is shown in FIG. 1(C). The sevenunderlined sequences show potential hydrophobic transmembrane domains.Shaded residues represent potential phosphorylation sites; PKA(R-X1-2-S/T-X) or PKC (X-S/T-X-R/K).

Example 2 Expression of R-14 mRNA in Human Trabecular Meshwork Tissue

[0228] Method: RT-PCR of total mRNA isolated from human trabecularmeshwork (HTM) and human iris (HI) tissues. Aliquots of total RNA (1 μg)were reverse transcribed (50° C. for 30 min) and the reaction mixturewas amplified for 35 cycles (60° C. for 30 sec) using gene-specificprimers SuperScript One-Step RT-PCR (InVitrogen). Resolution of the PCRproducts by agarose gel electrophoresis showed a single DNA fragment of1 Kb from HTM tissue, but not from iris tissue. Sequencing of the DNAfragment from HTM tissue identified the 1 Kb fragment to contain theR-14 reading frame.

Example 3 Expression of Cloned Human R-14 Receptor in Human Cells

[0229] Cell culture: HEK293 cells were grown in complete DMEM (10% fetalcalf serum, 0.1% Penicillin and streptomycin, 2 mM glutamine, 0.5 ug/mlfungizone, 5 ug/ml gentamicin) until ˜70% confluent. Cells weretransfected using Lipofectamine 2000 (InVitrogen) according to themanufacturer's recommendations using 12.5 μg DNA and 25 μl Lipofectaminein DMEM. Media was replaced after 48 hours for complete DMEM (The cellswere processed immediately to detect transient expression of R-14),supplemented with 500 μg/ml Zeocin. After 3 weeks in culture, thezeocin-resistant cells (R14/293) were split and cultured in completeDMEM with 100 μg/ml Zeocin.

[0230] Immunoblotting of R-14 proteins: Confluent cells were lysed inTNE (10 mM Tris-HCl pH 7.4, 0.1 mM EDTA, 0.85% NaCl, 1% NP-40) buffercontaining protease inhibitor cocktail. Aliquots (100 μg protein) ofsupernatants were denatured in 50 μl of SDS-loading buffer by boilingfor 5 minutes and resolved on 10% SDS-acrylamide gels. Proteins weretransferred to PVDF membrane by electroblotting and was blocked with 5%dry milk BLOTTO for 1 h, and incubated with mouse anti-HIS ({fraction(1/2000)}) for 1 h, washed, then incubated with goat anti mouse-HRP({fraction (1/2000)}) in TBST for 1 hour. Membrane was rinsed with TBSand developed by colorimetry using a DAB/CN substrate (Pierce). Thedetails of immunoblotting are given in Moore D et al (Ed) Currentprotocols in molecular biology 1987. John Wiley & sons inc. Withreference to FIG. 3, R14+ refer to HEK 293 cells expressing recombinantR14 fusion protein (HIS-tag) whereas R14− refer to parent HEK293 cellswhich do not contain native R-14 receptor. The immunoreactive R-14 bandis indicated by an arrow.

Example 4 Identification and Characterization of Peptide Inhibitors ofR-14 Protein

[0231] Method: Newborn pigs (1-3 days old) were anesthetized with 1.5%halothane for tracheostomy and catheterization of the right femoral veinfor drug administration. Animals were ventilated by means of a Harvardsmall animal respirator with a gas mixture of 25% 02 and 75% N₂.Halothane was discontinued after surgery and immediately thereafter theanimals were sedated with Alpha-chloralose (50 mg/kg i.v.) and paralyzedwith pancuronium (0.1 mg/kg i.v.). Animals were placed under radiantwarmer to keep their body temperature at 37° C. A butterfly needle (24G)is inserted into the anterior chamber of both eyes and connected to aStatham pressure transducer connected to a Gould multichannel recorder.Intraocular pressure is then allowed to stabilize for 15 minutes.Peptides were prepared by standard chemical methods of peptidesynthesis. Peptides dissolved in saline were topically applied as dropsunder the lower eyelid and allowed to diffuse. The intraocular pressurewas monitored for 15 minutes at which time a maximal effect wasobserved.

[0232] (A) Effects of peptides on intraocular pressure in piglet.Peptides dissolved in saline were applied to the eye after 15 min ofstabilization of intraocular pressure and changes in pressure weremonitored for 15 min. The deviation (negative for hypotension) wasplotted as a function of time (min). Of the peptides tested, 1401, 1402and 1405 produced a decrease in ocular pressure within minutes ofapplication, whereas 1406 and 1407 did not affect the basal IOP in theanimals.

[0233] (B) Dose-response of 1402 and 1405 peptides on intra-ocularpressure in piglets. The experiments were done as above, except thatintraocular pressure was plotted versus the dose of peptide used. Doseresponse of the peptides on basal IOP of piglet revealed IC50 values of86.4 and 341.6 nM for 1402 and 1405 respectively. TABLE 4 Alternativepeptides according to embodiments of the invention. Peptide SEQ ID NO:Sequence 1401 3 F S L T Q K Y C 1402 4 H I I C S P L R 1405 5 I F D S TE C W X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈

Example 5 Comparison of the Efficacy of 1405 with Latanoprost andTimolol

[0234] Method: Immediately after euthanasia, rabbit eyeballs werecollected and placed into 15-ml organ baths (Radnoti Glass, Monrovia,Calif.). Baths were filled with Krebs (composition in mM: NaCl 120, KCl4.5, CaCl₂ 2.5, MgSO₄ 1.0, NaHCO₃ 27, KH₂PO₄ 1.0, and glucose 10, pH7.4), maintained at 37° C. and bubbled with 95% 02. The eyeballs weremaintained in place with the cornea facing upwards. A butterfly needle(24G) is inserted into the anterior chamber of the eye and connected toa Statham pressure transducer connected to a Gould multichannelrecorder. Intraocular pressure is then increased to 35 mmHg manuallywith saline. The recovery rate (return to 20 mmHg) in the absence(control) or presence of topically applied agents was assessed. Twomeasurements were averaged, both in the relatively linear portion of therecovery. Control measurements were repeated twice, giving identicalresults. All values were obtained on the same rabbit eye. Ophthalmicpreparations of latanoprost (0.03%) and timolol (0.5%) in addition topeptide 1405 (0.1%) in saline were applied as drops to the eyes. Peptide1405 reversed experimentally-induced ocular hypertension with anefficiency similar to that of timolol, but significantly faster thanlatanoprost.

Example 6 Selectivity of Peptides 1402 and 1405

[0235] Method: Adult pig eyecup preparations are used to study theresponse in situ of the relatively undisturbed retinal vasculature.Briefly, a circular incision is made 3-4 mm posterior to the ora serratato remove the anterior segment and vitreous body with minimal handlingof the retina. The remaining eyecup is fixed with pins to a wax base ina 20-ml tissue bath containing Krebs buffer (pH 7.35-7.45) equilibratedwith 21% 02 and 5% CO₂ and maintained at 37° C. The preparations areallowed to stabilize for 30-45 min, during which they were rinsed two orthree times with fresh buffer. Cumulative concentration-response curvesto different agents are constructed separately on nonperfused primaryarterioles (100-200 μm diameter) of fresh tissue. The outer vesseldiameter is recorded with a video camera mounted on a dissectingmicroscope (model M-400, Zeiss), and responses are quantified by adigital image analyzer (Sigma Scan software, Jandel Scientific, CorteMadera, Calif.). Vascular diameter is recorded before and 10 min aftertopical application of each concentration of agent, at which time astable response is generally achieved. Each measurement is repeatedthree times, and variability is <1%. Additional experiments can beperformed after a 20 min pretreatment with a variety of blocking ormodulating agents. The responses are expressed as percent change in theouter diameter of vessel from baseline or as a percent reversal of aconstrictor agent (Thromboxane receptor agonist, U46619 at aconcentration producing a 70% of its maximal effects).

[0236] Both latanoprost and timolol constricted the porcine adultretinal arterioles by an average of 7-8% (FIG. 6A). The R-14 antagonistpeptides 1402 and 1405 were tested to measure the relative selectivityof these compounds compared to known ocular hypotensive compounds,latanoprost and timolol. Both 1402 and 1405 peptides did not reverse theconstriction produced by latanoprost (by 1-5%) or by timolol (<1%).

Example 7 Effect of Peptides 1402 (A) and 1405 (B) on IntraocularPressure in Rabbits

[0237] New Zealand white rabbits were trained to stay calm during theapplication of eye drops and measurement of intraocular pressure (IOP)by flourescein tonometry. Peptides were freshly made in phosphatebuffered saline at concentrations indicated and a drop (50 μl) wasapplied in the irido-corneal angle of the eye. The IOP measurements weretaken at different times. As shown in FIG. 7, there was an immediatedecrease in IOP by the application of either peptide and the IOPreturned to normal values within 4 h. Repeated application of thepeptide produced similar decreases in IOP and the effects are reversiblewith the disappearance of the peptides.

[0238] Throughout this application, various references are referred todescribe more fully the state of the art to which this inventionpertains. The disclosures of these references are hereby incorporated byreference into the present disclosure.

1 8 1 969 DNA Homo sapiens CDS (1)..(969) coding sequence forpolypeptide 1 atg gat tca acc atc cca gtc ttg ggt aca gaa ctg aca ccaatc aac 48 Met Asp Ser Thr Ile Pro Val Leu Gly Thr Glu Leu Thr Pro IleAsn 1 5 10 15 gga cgt gag gag act cct tgc tac aag cag acc ctg agc ttcacg ggg 96 Gly Arg Glu Glu Thr Pro Cys Tyr Lys Gln Thr Leu Ser Phe ThrGly 20 25 30 ctg acg tgc atc gtt tcc ctt gtc gcg ctg aca gga gac gcg gttgtg 144 Leu Thr Cys Ile Val Ser Leu Val Ala Leu Thr Gly Asp Ala Val Val35 40 45 ctc tgg ctc ctg ggc tgc cgc atg cgc agg aac gct gtc tcc atc tac192 Leu Trp Leu Leu Gly Cys Arg Met Arg Arg Asn Ala Val Ser Ile Tyr 5055 60 atc ctc aac ctg gtc gcg gcc gac ttc ctc ttc ctt agc ggc cac att240 Ile Leu Asn Leu Val Ala Ala Asp Phe Leu Phe Leu Ser Gly His Ile 6570 75 80 ata tgt tcg ccg tta cgc ctc atc aat atc cgc cat ccc atc tcc aaa288 Ile Cys Ser Pro Leu Arg Leu Ile Asn Ile Arg His Pro Ile Ser Lys 8590 95 atc ctc agt cct gtg atg acc ttt ccc tac ttt ata ggc cta agc atg336 Ile Leu Ser Pro Val Met Thr Phe Pro Tyr Phe Ile Gly Leu Ser Met 100105 110 ctg agc gcc atc agc acc gag cgc tgc ctg tcc atc ctg tgg ccc atc384 Leu Ser Ala Ile Ser Thr Glu Arg Cys Leu Ser Ile Leu Trp Pro Ile 115120 125 tgg tac cac tgc cgc cgc ccc aga tac ctg tca tcg gtc atg tgt gtc432 Trp Tyr His Cys Arg Arg Pro Arg Tyr Leu Ser Ser Val Met Cys Val 130135 140 ctg ctc tgg gcc ctg tcc ctg ctg cgg agt atc ctg gag tgg atg ttc480 Leu Leu Trp Ala Leu Ser Leu Leu Arg Ser Ile Leu Glu Trp Met Phe 145150 155 160 tgt gac ttc ctg ttt agt ggt gct gat tct gtt tgg tgt gaa acgtca 528 Cys Asp Phe Leu Phe Ser Gly Ala Asp Ser Val Trp Cys Glu Thr Ser165 170 175 gat ttc att aca atc gcg tgg ctg gtt ttt tta tgt gtg gtt ctctgt 576 Asp Phe Ile Thr Ile Ala Trp Leu Val Phe Leu Cys Val Val Leu Cys180 185 190 ggg tcc agc ctg gtc ctg ctg gtc agg att ctc tgt gga tcc cggaag 624 Gly Ser Ser Leu Val Leu Leu Val Arg Ile Leu Cys Gly Ser Arg Lys195 200 205 atg ccg ctg acc agg ctg tac gtg acc atc ctc ctc aca gtg ctggtc 672 Met Pro Leu Thr Arg Leu Tyr Val Thr Ile Leu Leu Thr Val Leu Val210 215 220 ttc ctc ctc tgt ggc ctg ccc ttt ggc att cag tgg gcc ctg ttttcc 720 Phe Leu Leu Cys Gly Leu Pro Phe Gly Ile Gln Trp Ala Leu Phe Ser225 230 235 240 agg atc cac ctg gat tgg aaa gtc tta ttt tgt cat gtg catcta gtt 768 Arg Ile His Leu Asp Trp Lys Val Leu Phe Cys His Val His LeuVal 245 250 255 tcc att ttc ctg tcc gct ctt aac agc agt gcc aac ccc atcatt tac 816 Ser Ile Phe Leu Ser Ala Leu Asn Ser Ser Ala Asn Pro Ile IleTyr 260 265 270 ttc ttc gtg ggc tcc ttt agg cag cgt caa aat agg cag aacctg aag 864 Phe Phe Val Gly Ser Phe Arg Gln Arg Gln Asn Arg Gln Asn LeuLys 275 280 285 ctg gtt ctc cag agg gct ctg cag gac acg cct gag gtg gatgaa ggt 912 Leu Val Leu Gln Arg Ala Leu Gln Asp Thr Pro Glu Val Asp GluGly 290 295 300 gga ggg tgg ctt cct cag gaa acc ctg gag ctg tcg gga agcaga ttg 960 Gly Gly Trp Leu Pro Gln Glu Thr Leu Glu Leu Ser Gly Ser ArgLeu 305 310 315 320 gag cag taa 969 Glu Gln 2 322 PRT Homo sapiens 2 MetAsp Ser Thr Ile Pro Val Leu Gly Thr Glu Leu Thr Pro Ile Asn 1 5 10 15Gly Arg Glu Glu Thr Pro Cys Tyr Lys Gln Thr Leu Ser Phe Thr Gly 20 25 30Leu Thr Cys Ile Val Ser Leu Val Ala Leu Thr Gly Asp Ala Val Val 35 40 45Leu Trp Leu Leu Gly Cys Arg Met Arg Arg Asn Ala Val Ser Ile Tyr 50 55 60Ile Leu Asn Leu Val Ala Ala Asp Phe Leu Phe Leu Ser Gly His Ile 65 70 7580 Ile Cys Ser Pro Leu Arg Leu Ile Asn Ile Arg His Pro Ile Ser Lys 85 9095 Ile Leu Ser Pro Val Met Thr Phe Pro Tyr Phe Ile Gly Leu Ser Met 100105 110 Leu Ser Ala Ile Ser Thr Glu Arg Cys Leu Ser Ile Leu Trp Pro Ile115 120 125 Trp Tyr His Cys Arg Arg Pro Arg Tyr Leu Ser Ser Val Met CysVal 130 135 140 Leu Leu Trp Ala Leu Ser Leu Leu Arg Ser Ile Leu Glu TrpMet Phe 145 150 155 160 Cys Asp Phe Leu Phe Ser Gly Ala Asp Ser Val TrpCys Glu Thr Ser 165 170 175 Asp Phe Ile Thr Ile Ala Trp Leu Val Phe LeuCys Val Val Leu Cys 180 185 190 Gly Ser Ser Leu Val Leu Leu Val Arg IleLeu Cys Gly Ser Arg Lys 195 200 205 Met Pro Leu Thr Arg Leu Tyr Val ThrIle Leu Leu Thr Val Leu Val 210 215 220 Phe Leu Leu Cys Gly Leu Pro PheGly Ile Gln Trp Ala Leu Phe Ser 225 230 235 240 Arg Ile His Leu Asp TrpLys Val Leu Phe Cys His Val His Leu Val 245 250 255 Ser Ile Phe Leu SerAla Leu Asn Ser Ser Ala Asn Pro Ile Ile Tyr 260 265 270 Phe Phe Val GlySer Phe Arg Gln Arg Gln Asn Arg Gln Asn Leu Lys 275 280 285 Leu Val LeuGln Arg Ala Leu Gln Asp Thr Pro Glu Val Asp Glu Gly 290 295 300 Gly GlyTrp Leu Pro Gln Glu Thr Leu Glu Leu Ser Gly Ser Arg Leu 305 310 315 320Glu Gln 3 8 PRT Artificial sequence synthetic peptide 3 Phe Ser Leu ThrGln Lys Tyr Cys 1 5 4 8 PRT Artificial sequence synthetic peptide 4 HisIle Ile Cys Ser Pro Leu Arg 1 5 5 8 PRT Artificial sequence syntheticpeptide 5 Ile Phe Asp Ser Thr Glu Cys Trp 1 5 6 28 DNA Artificialsequence derived from Homo sapiens 6 gattcaacca tcccagtctt gggtacag 28 728 DNA Artificial sequence derived from Homo sapiens 7 ttactgctccaatctgcttc ccgacagc 28 8 19 DNA Artificial sequence derived from vectorpcDNA4HisMax-TOPO 8 tatggctagc atgactggt 19

What is claimed is:
 1. A substantially pure peptide compound of FormulaI: Z₁-X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈-Z₂  I wherein: X₁ is selected from thegroup consisting of Phe, His, Ile and related amino acids; X₂ isselected from the group consisting of Ser, Ile, Phe and related aminoacids X₃ is selected from the group consisting of Leu, Ile, Asp andrelated amino acids X₄ is selected from the group consisting of Thr,Cys, Ser and related amino acids possessing side chains containingsulfhydryl, hydroxyl or H-bond forming groups; X₅ is selected from thegroup consisting of Gln, Ser, Thr and related amino acids; X₆ isselected from the group consisting of Lys, Pro, Glu and related aminoacids; X₇ is selected from the group consisting of Tyr, Leu, Cys andrelated amino acids; X₈ is selected from the group consisting of Cys,Arg, Trp and related amino acids; Z₁ is an N-terminal group of theformula H₂N—, RHN— or, RRN—; Z₂ is a C-terminal group of the formula—C(O)OH, —C(O)R, —C(O)OR, —C(O)NHR, —C(O)NRR; R at each occurrence isindependently selected from (C₁-C₆) alkyl, (C₁-C₆) alkenyl, (C₁-C₆)alkynyl, substituted (C₁-C₆) alkyl, substituted (C₁-C₆) alkenyl, orsubstituted (C₁-C₆) alkynyl; and “-” is a covalent linkage.
 2. Asubstantially pure synthetic peptide compound or recombinant peptidecompound, said compound having a domain of Formula II:—X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈  II wherein: X₁ is selected from the groupconsisting of Phe, His, Ile and related amino acids; X₂ is selected fromthe group consisting of Ser, Ile, Phe and related amino acids X₃ isselected from the group consisting of Leu, Ile, Asp and related aminoacids X₄ is selected from the group consisting of Thr, Cys, Ser andrelated amino acids possessing side chains containing sulfhydryl,hydroxyl or H-bond forming groups; X₅ is selected from the groupconsisting of Gln, Ser, Thr and related amino acids; X₆ is selected fromthe group consisting of Lys, Pro, Glu and related amino acids; X₇ isselected from the group consisting of Tyr, Leu, Cys and related aminoacids; X₈ is selected from the group consisting of Cys, Arg, Trp andrelated amino acids; “-” is a covalent linkage.
 3. The compound of claim1, wherein said compound is selected from the group consisting of:FSLTQKYC; HIICSPLR; and IFDSTECW.
 4. The compound of claim 2, whereinsaid domain is selected from the group consisting of: FSLTQKYC;HIICSPLR; and IFDSTECW.
 5. A method of lowering intraocular pressure ina subject, said method comprising administering to said subject aneffective amount of the compound of claim 1
 6. A method of treating in asubject a condition associated with elevated intraocular pressure, saidmethod comprising administering to said subject an effective amount ofthe compound of claim
 1. 7. The method of claim 6, wherein the conditionis glaucoma.
 8. The method of claim 5, wherein said method comprisesadministering said compound to an eye of said subject.
 9. The method ofclaim 5, wherein the subject is a mammal.
 10. The method of claim 9,wherein the subject is human.
 11. A pharmaceutical compositioncomprising the compound of claim 1 in admixture with a pharmaceuticallyacceptable carrier.
 12. The pharmaceutical composition of claim 11,wherein said composition is formulated for administration to an eye of asubject.
 13. A commercial package comprising the compound of claim 1together with instructions for its use.
 14. The commercial package ofclaim 13, wherein said use is selected from the group consisting of: (a)lowering intraocular pressure in a subject; and (b) treating a conditionassociated with elevated intraocular pressure.
 15. The commercialpackage of claim 14, wherein said condition is glaucoma.
 16. An isolatednucleic acid comprising a sequence that encodes the domain of claim 2.17. A vector comprising the nucleic acid of claim 16 operably linked toa transcriptional regulatory element.
 18. A host cell comprising thevector of claim
 17. 19. A method of producing the peptide compound ofclaim 2, comprising culturing the host cell of claim 18 under conditionspermitting expression of the peptide compound.
 20. An isolated nucleicacid comprising a sequence that encodes a polypeptide comprising atleast 278 amino acids of SEQ ID NO:2.
 21. The isolated nucleic acid ofclaim 20, wherein said polypeptide comprises SEQ ID NO:2.
 22. Theisolated nucleic acid of claim 21, wherein said nucleic acid comprisesSEQ ID NO:1.
 23. A substantially pure polypeptide comprising at least278 consecutive amino acids of SEQ ID NO:2.
 24. The polypeptide of claim23, said polypeptide comprising SEQ ID NO:2.
 25. A vector comprising thenucleic acid of claim 20 operably linked to a transcriptional regulatoryelement.
 26. A host cell comprising the vector of claim
 25. 27. A methodof producing the polypeptide of claim 23, comprising culturing the hostcell of claim 26 under conditions permitting the expression of thepolypeptide.
 28. A method for identifying a compound for: (i) loweringintraocular pressure in a subject; (ii) treating a condition associatedwith elevated intraocular pressure; or (iii) both (i) and (ii); saidmethod comprising: (a) providing a cell expressing an R-14; (b)contacting the cell with a test compound; and (c) determining whetherR-14 activity is decreased in the presence of said test compound, saiddecrease in activity being an indication that said compound may beuseful for: (i) lowering intraocular pressure in a subject; (ii)treating a condition associated with elevated intraocular pressure; or(iii) both (i) and (ii).
 29. The method of claim 28 wherein saiddecrease in activity is an indication that said compound is an R-14antagonist.
 30. The method of claim 28, wherein said R-14 comprises apolypeptide comprising SEQ ID NO:2, an active fragment thereof, or asequence substantially identical thereto.